CN120775060A

CN120775060A - TACI/BCMA chimeric fusion proteins and uses thereof

Info

Publication number: CN120775060A
Application number: CN202510399567.9A
Authority: CN
Inventors: 熊尧; 周帅祥; 张志敏
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2024-04-02
Filing date: 2025-04-01
Publication date: 2025-10-14
Also published as: WO2025209465A1

Abstract

本发明涉及一种新的TACI/BCMA嵌合体，以及包含其的融合蛋白。本发明还涉及编码所述嵌合体或融合蛋白的核酸、包含所述核酸的载体，以及包含所述核酸或载体的宿主细胞。本发明还涉及所述融合蛋白用于治疗免疫相关疾病的治疗方法和用途。The present invention relates to a novel TACI/BCMA chimera and fusion proteins comprising the same. The present invention also relates to nucleic acids encoding the chimera or fusion protein, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. The present invention also relates to therapeutic methods and uses of the fusion proteins for treating immune-related diseases.

Description

TACI/BCMA chimeric fusion proteins and uses thereof

Cross Reference to Related Applications

The present application is based on chinese patent application No. 202410396507.7, application No. 2024, 4, 2, and application No. 20241500688. X, application No. 2024, 10, 25, and application No. 202510355955.7, application No. 2025, 3, 25, and claims priority from the three chinese patent applications, all of which are incorporated herein by reference.

The present invention relates to a novel TACI/BCMA chimera, and fusion proteins comprising the same. The invention also relates to nucleic acids encoding said chimeric or fusion proteins, vectors comprising said nucleic acids, and host cells comprising said nucleic acids or vectors. The invention also relates to therapeutic methods and uses of the fusion proteins for the treatment of immune related diseases.

Background

The cytokine Tumor Necrosis Factor (TNF) was reported in 1968 as a cytotoxic factor that induces tumor necrosis. The protein is produced as a type II transmembrane protein 233 amino acids long and is released in the extracellular space as a soluble homotrimer by proteolytic cleavage by the metalloprotease TNF-alpha converting enzyme (TACE/ADAM 17). TNF- α (TNFSF 1B) and TNF- β (TNFSF 1A) bind to two specific receptors (TNFR 1 and TNFR 2).

The TNF superfamily (TNFSF) now includes 19 molecules, while the TNF receptor superfamily (TNFRSF) includes 29 distinct protein members. In addition to the one-to-one selectivity between specific ligands and receptors, cross-reactivity was also reported, suggesting differential signaling and a variety of putative cellular and molecular roles.

TNF receptor superfamily (TNFRSF) refers to a group of cell surface cytokine receptors, all of which are type I (N-terminal extracellular) transmembrane glycoproteins that contain one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of this superfamily based on shared structural features including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular regions, which typically play a role in the binding of proteins to their cognate binding partners or ligands. TNFRSF protein may have only one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or extracellular domain of a TNFRSF member contains 1 to 6 CRD pseudo-repeat sequences. For example, BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD 1 and CRD 2). TNFRSF members are typically trimeric or multimeric complexes that are stabilized by disulfide bonds within their cysteines. Binding of TNFRSF protein to its ligand promotes various biological activities in cells, such as apoptotic cell death or induction of cell survival and proliferation.

BAFF (B CELL ACTIVATING factor) and APRIL (Aproliferation-inducing ligand) are B cell activating and regulating factors belonging to TNF family, can promote the development and proliferation of B cells, promote the secretion of various immunoglobulins of organism, increase the expression level in serum, and have important regulating effect on immune response of organism. They bind to the cell membrane receptors TACI (Transmembrane activator andCAML-interactor) and BCMA (B cell maturation antigen). In addition, BAFF is also able to bind to another receptor BAFFR (B cell-ACTIVATING FACTOR RECEPTOR). BAFF and APRIL regulate lymphocyte activation, development and proliferation via signaling by these several receptors. Overexpression of BAFF and APRIL is one of the etiologies of a variety of autoimmune diseases. The study shows that the serum BAFF and APRIL concentration of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and the like are obviously increased. Targeting BAFF/APRIL is therefore an effective approach to the treatment of autoimmune diseases.

Transmembrane activator and CAML interacting protein (TACI), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF 13B), were originally discovered because of their ability to interact with calcium modulators and cyclophilin ligands (CAMLs). TACI was later found to play a critical role in humoral immunity by interacting with two members of the TNF family, BAFF and APRIL.

The prior art has found fusion proteins comprising TACI for the treatment of immune disorders. For example Rong Chang biology produced an optimised TACI-Fc fusion protein, tai-cit-p (CN 101323643B), which was modified with the full-length TACI extracellular domain, but the final drug molecule stability was poor, the final formulation was only in lyophilized powder form, and the clinical dosing cycle was 1 week 2 needles, and the dosing frequency could not be achieved by the strategy of increasing the dosing amount.

Thus, there remains a need in the art to develop a highly drug-resistant, molecularly stable TACI fusion protein.

Disclosure of Invention

The present invention relates to TACI/BCMA chimeras, and fusion proteins comprising the same, e.g., with an Fc region.

The Fc region fusion proteins of the TACI/BCMA chimeras of the present invention have better stability, e.g., thermostability, than known TACI-Fc fusion proteins, while also having a longer half-life and/or lower clearance.

In some embodiments, the invention relates to a TACI/BCMA chimera comprising a chimeric protein that uses the N-terminus and/or C-terminus of BCMA to replace the N-terminus and/or C-terminus of a functional fragment of the TACI extracellular domain ECD.

In a specific embodiment, the TACI/BCMA chimera comprises the structure:

BCMAN terminal amino acid-TACI moiety, wherein the TACI moiety is an extracellular domain ECD or a functional fragment thereof, or

BCMAN terminal amino acid-TACI portion-BCMAC terminal amino acid, wherein the TACI portion is the extracellular domain ECD of TACI deleted of the N-and C-termini or a functional fragment thereof.

In a specific embodiment, the extracellular domain ECD functional fragment of TACI in the TACI/BCMA chimera comprises CRD2 of TACI and does not comprise CRD1 or any fragment of CRD 1. In one embodiment, the CRD2 is TACI corresponding to the amino acid sequence at positions 71-104 shown in SEQ ID NO. 1, and CRD1 is TACI corresponding to the amino acid sequence at positions 34-66 shown in SEQ ID NO. 1. In one embodiment, the extracellular domain ECD functional fragment of TACI further comprises a partial stem region of TACI and/or the TACI corresponds to any amino acid sequence between positions 68-70 of SEQ ID NO. 1. In one embodiment, the extracellular domain ECD functional fragment of TACI is or comprises a fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO. 1, amino acid residues 68-110. In one embodiment, the extracellular domain ECD functional fragment of TACI comprises

(I) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 31, 32 or 33;

(ii) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 33 and having Y102D;

(iii) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 32 and having K77E, F Y and Y102D;

(iv) The amino acid sequence shown in SEQ ID NO 31, 32 or 33, or

(V) Consisting of a sequence according to any one of (i) - (iv).

In a specific embodiment, the N-terminus of the extracellular domain ECD functional fragment of TACI refers to the N-terminal amino acid of TACI before the amino acid corresponding to position Y79 of SEQ ID NO. 1.

In a specific embodiment, the C-terminal end of the extracellular domain ECD functional fragment of TACI means that the TACI corresponds to the C-terminal amino acid after amino acid 99, amino acid 100, amino acid 101, amino acid 102, amino acid 103, amino acid 104, amino acid 105, amino acid 106, amino acid 107, amino acid 108, amino acid 109 or amino acid 110, preferably the C-terminal amino acid after amino acid 99 or amino acid 105 of SEQ ID NO. 1.

In a specific embodiment, the N-terminal amino acid of BCMA is selected from the group consisting of amino acid sequences of BCMA corresponding to positions 1-13, positions 2-13, positions 3-13, positions 4-13, positions 5-13, positions 6-13, or positions 7-13 of SEQ ID NO. 2. In a specific embodiment, the N-terminal amino acid sequence of BCMA comprises, or consists of, the amino acid sequence set forth in any one of SEQ ID NOs 40-46.

In a specific embodiment, the C-terminal amino acid sequence of BCMA is the amino acid sequence of BCMA corresponding to positions 37-47, 37-46, 37-45, 37-44 of SEQ ID NO. 2, or the amino acid sequence corresponding to positions 43-44 or 43-46 of SEQ ID NO. 2.

In a specific embodiment, the C-terminal amino acid of BCMA comprises a mutation, e.g., a substitution, e.g., N42A or N42Q or R39D or N42A-R39D, at position 39 and/or 42 to improve binding affinity, stability, and/or drug resistance.

In a specific embodiment, the C-terminal amino acid sequence of BCMA comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs 47-55.

In a specific embodiment, the TACI ECD functional fragment comprises an amino acid site mutation that reduces the risk of aggregation, e.g. a mutation such as a substitution at a pharmaceutical risk site, e.g. selected from amino acids 69, 72, 73, 74, 77, 78, 85, 102 or 103 corresponding to SEQ ID No.1, preferably the amino acid of the pharmaceutical risk site is mutated to a or D, e.g. the mutation is Y102D.

In a specific embodiment, the TACI portion comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs 31-38, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity thereto.

In a specific embodiment, the TACI/BCMA chimera comprises, or consists of, an amino acid sequence set forth in any one of SEQ ID NOs 59-80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity thereto.

In one aspect, the invention relates to a fusion protein comprising a TACI/BCMA chimera as described herein, and an Fc region.

In a specific embodiment, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

In a specific embodiment, the Fc region comprises one or more of the following mutations:

(i) Deletion of lysine K (K447 del) at the C-terminus;

(ii) Mutations that reduce effector function mediated by the Fc region;

(iii) Mutations that reduce binding to the Fcγ receptor, such as the L234A/L235A mutation or the L234A/L235E/G237A mutation;

(iv) Mutations that enhance binding of the Fc fragment to FcRn, such as M252Y/S254T/T256E and/or M482L/N434S.

In a specific embodiment, the Fc region comprises

(I) 81 or 82 or an amino acid sequence having at least 90% identity thereto, e.g., 95%,96%,97%,99% or more identity thereto;

(ii) 83 or 84, or an amino acid sequence having at least 90% identity thereto, e.g., 95%,96%,97%,99% or more identity thereto, optionally deleted for a C-terminal lysine;

(iii) The amino acid sequence set forth in any one of SEQ ID NOs 85-88 and 100-103 or an amino acid sequence having at least 90% identity thereto, e.g., 95%,96%,97%,98%,99% or more identity;

(iv) An amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 85 or 87 and comprising the mutation L234A/L235E/G237A;

(v) Amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 86 or 88 and comprising the mutations L234A/L235E/G237A and C-terminally deleted lysines;

(vi) Amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 100 or 102 and comprising the mutations L234A/L235E/G237A and M252Y/S254T/T256E;

(vii) An amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO 101 or 103 and comprising the mutations L234A/L235E/G237A, M252Y/S254T/T256E and C-terminal deleted lysine, or

(Viii) Consists of the amino acid sequence of any one of the above (i) - (vii).

In a specific embodiment, the TACI/BCMA chimera is fused to the Fc directly or through a linker, preferably the C-terminus of the chimera is fused to the N-terminus of the Fc directly or through a linker. In a specific embodiment, the linker is selected from (GSGGGGS) _n、(GS)_n、(GSGGS)_n、(GGGGS)_n or (GGGS) _n, wherein n is an integer of at least 1, such as 1,2,3, 4 or 5, such as (GSGGGGS) _n, wherein n=1-3, such as the linker is the amino acid sequence depicted in SEQ ID NO: 39.

In a specific embodiment, the fusion protein

(I) Comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of any one of SEQ ID NOs 5 to 28, or

(Ii) Comprising or consisting of the amino acid sequence of any one of SEQ ID NOs 5 to 28

In one aspect, the invention relates to a fusion protein dimer comprising a first monomer and a second monomer, said first and second monomer comprising or consisting of a fusion protein chain as described herein, respectively, preferably the first and second monomer are identical.

In one aspect, the invention relates to a polynucleotide encoding a TACI/BCMA chimeric or fusion protein dimer of the invention.

In one aspect, the invention relates to an expression vector comprising a polynucleotide of the invention, e.g., the expression vector is a pCDNA expression vector, e.g., a pCDNA3.1 expression vector.

In one aspect, the invention relates to a host cell comprising a polynucleotide or expression vector of the invention.

In one aspect, the present invention relates to a method of making a TACI/BCMA chimera or fusion protein thereof or fusion protein dimer, wherein said method comprises culturing a host cell of the present invention under conditions suitable for expression of the TACI/BCMA chimera or fusion protein thereof or fusion protein dimer, and optionally recovering said TACI/BCMA chimera or fusion protein thereof or fusion protein dimer from said host cell (or host cell culture medium).

In one aspect, the invention relates to a pharmaceutical composition comprising a TACI/BCMA chimera or fusion protein dimer of the invention, and optionally a pharmaceutical excipient.

In one aspect, the invention relates to a pharmaceutical combination or combination product comprising a TACI/BCMA chimeric or fusion protein dimer of the invention, and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents affecting T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).

In one aspect, the invention relates to a method of preventing or treating a disease, such as a B cell or autoantibody related disease or an immune system disease (e.g. autoimmune disease) or inflammation in a subject, comprising administering to the subject a TACI/BCMA chimeric or fusion protein dimer or pharmaceutical composition or combination of the invention. In a specific embodiment, the B cell or autoantibody related disease is a B cell or autoantibody mediated disease, e.g., a B cell or autoantibody mediated autoimmune disease. In a specific embodiment, the B cell or autoantibody related disease or immune system disease or inflammation is a disease (e.g., autoimmune disease) in which B cells proliferate abnormally or activate abnormally in a subject compared to a sample of a healthy subject. In a specific embodiment, the disease is lupus, e.g., systemic lupus erythematosus, rheumatoid arthritis, igA nephropathy (IgAN) or membranous nephropathy, chronic kidney disease such as Sjogren's syndrome, myasthenia gravis, idiopathic Thrombocytopenic Purpura (ITP), warm-blooded autoimmune hemolytic anemia (wAIHA), multiple Sclerosis (MS), coronary heart disease (CAD), or thyroid-eye disease. In a specific embodiment, the administration further comprises co-administration of one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatics affecting T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).

Drawings

FIG. 1 shows structural analysis of the formation of complexes of TACI and BCMA with APRIL, respectively.

Fig. 2 shows structural alignment and analysis of TACI and BCMA.

FIG. 3 shows amino acid sequence alignment of TACI with BCMA.

FIG. 4 shows the ability of TACI/BCMA chimeric fusion proteins to inhibit B cell proliferation.

FIG. 5 shows that TACI/BCMA chimeric fusion proteins inhibit proliferation of mouse spleen cells and spleen B cells resulting from KLH immunization.

FIG. 6 shows that TACI/BCMA chimeric fusion proteins inhibit the levels of IgA, igM and IgG in serum of mice induced by KLH immunization.

FIG. 7 shows the pharmacokinetics of TACI/BCMA chimeric fusion proteins in mice.

FIG. 8 shows the toxicity kinetics of TACI/BCMA chimeric fusion proteins in cynomolgus monkeys.

FIG. 9 shows the pharmacokinetics of TACI/BCMA chimeric fusion proteins and YTE molecules thereof in cynomolgus monkeys.

FIG. 10 shows immunoglobulin level changes in cynomolgus monkey serum for TACI/BCMA chimeric fusion proteins and YTE molecules thereof.

Detailed Description

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

I. Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5%, 4%, 3%, 2%, or 1% less than the specified numerical value and an upper limit of 5%, 4%, 3%, 2%, or 1% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the selectable items or two or more of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to "comprising" or "including" a certain chimera, it is also intended to cover having only that chimera.

In this context, parental TACI refers to a template for introducing a mutation of the invention, which may be a wild-type TACI, e.g., a naturally occurring TACI protein, e.g., a naturally occurring TACI from human, mouse, rat, non-human primate, including unprocessed (e.g., signal peptide removed) and processed (e.g., N-terminal methionine removed) forms, or, e.g., naturally occurring TACI allelic variants and splice variants, isoforms, homologs, and species homologs, or, e.g., TACI variants, e.g., which may have at least 95%, 96%, 97%, 98% or 99% or more identity to the naturally occurring TACI or have no more than 1-10 or 1-5 amino acid mutations (e.g., conservative substitutions), and preferably have substantially the same BAFF binding affinity and/or APRIL binding affinity as the naturally occurring TACI protein. In one embodiment, the parent TACI refers to a functional fragment of TACI, e.g., a fragment comprising the extracellular domain (ECD) of TACI or the CRD2 domain of TACI, e.g., the extracellular domain (ECD) of TACI, or the CRD2 domain of TACI, or other functional fragment of TACI.

In the present invention, when referring to amino acid positions in a TACI protein or TACI sequence segment, it is determined by reference to the amino acid sequence SEQ ID No. 1 of a wild-type human TACI protein (also known as TACI ^WT). Corresponding amino acid positions on other TACI proteins or polypeptides (including full length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID No. 1. Thus, in the present invention, unless otherwise indicated, the amino acid positions of a TACI protein or polypeptide are the amino acid positions numbered according to SEQ ID No. 1. For example, when referring to "Y102" it refers to tyrosine residue Y at position 102 of SEQ ID NO. 1, or amino acid residues aligned at corresponding positions on other TACI polypeptide sequences.

In the present invention, when referring to amino acid positions in a BCMA protein or BCMA sequence section, it is determined by reference to the amino acid sequence SEQ ID No. 2 of a wild type human BCMA protein (also referred to as BCMA ^WT). Corresponding amino acid positions on other BCMA proteins or polypeptides (including full length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID No. 2. Thus, in the present invention, unless otherwise indicated, the amino acid positions of a BCMA protein or polypeptide are the amino acid positions numbered according to SEQ ID No. 2. For example, when referring to "Q3", it refers to tyrosine residue Q at position 3 of SEQ ID NO. 2, or an amino acid residue aligned at a corresponding position on the other TACI polypeptide sequences.

In this context, when referring to muteins, single amino acid substitutions are described in the following manner [ original amino acid residues/positions/substituted amino acid residues ]. For example, the tyrosine substitution at position 102 is aspartic acid, which can be represented as Y102D. When there are a number of alternative amino acid substitutions (e.g., D, E) at a given position (e.g., position Y102), the amino acid substitution can be expressed as Y102D/E. Accordingly, individual single amino acid substitutions may be joined by a plus sign "+" or "-" to represent a combined mutation at a plurality of given positions. For example, a combinatorial mutation at position K77E, F, Y, Y D may be expressed as K77E-F78Y-Y102D, or K77E+F78Y+Y102D.

As used herein, "N-terminal amino acid" or "N-terminal" are used interchangeably to refer to 1 or more amino acid segments starting at the very N-terminus of BCMA or TACI or a functional fragment thereof.

Herein, "C-terminal amino acid" or "C-terminal" are used interchangeably to refer to a1 amino acid or more than 1 amino acid segment ending at the very C-terminus of BCMA or TACI or a functional fragment thereof.

As used herein, the term "chimeric" refers to a fusion protein formed by splicing fragments of two or more different proteins by genetic engineering means. Such chimeras generally retain the original function of the individual fragments and may exhibit new biological properties or enhanced stability.

As used herein, the term "site of risk of pharmaceutical formation" refers to a site on a pharmaceutically acceptable protein, such as a chimeric or fusion protein of the invention, that affects its pharmaceutical properties (e.g., stability, etc.), including post-translational modification sites such as isomeriztion isomerisation (D), deamidation deamidation (N), glycosylationN glycosylation (N.S/T), free cysteine (C), oxidation (M/W), and enrichment regions (patch) of hydrophobic or charged amino acid residues over a larger exposed area of the protein surface, etc.

In this context, the "percent sequence identity" may be determined by comparing the two optimally aligned sequences over a comparison window. Preferably, sequence identity is determined over the full length of the reference sequence (e.g., SEQ ID NO: 1). Sequence alignment methods for comparison are well known in the art. Algorithms suitable for determining the percent sequence identity include, for example, BLAST and BLAST 2.0 algorithms (see Altschul et al, nuc. Acids Res.25:3389-402,1977 and Altschul et al J. Mol. Biol.215:403-10, 1990). Software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Center for Biotechnology Information). For the purposes of the present application, the percent identity is determined using the default parameters using Basic LocalAlignment Search Tool available from https:// blast.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to the substitution of one amino acid for another that has similar chemical properties (e.g., charge or hydrophobicity). Conservative substitution tables of functionally similar amino acids are well known in the art. In some embodiments, the following are exemplary conservative substitutions:

For example, the parent TACI protein may have conservative amino acid substitutions relative to one of SEQ ID nos. 1, or only conservative amino acid substitutions, and in a preferred embodiment, conservative substitutions are NO more than 10 amino acid residues, such as 1,2,3,4,5,6,7,8,9,10 residues. For another example, a mutant TACI protein of the invention may have conservative amino acid substitutions relative to the TACI mutant protein sequences specifically set forth herein, or only conservative amino acid substitutions and in a preferred embodiment, conservative substitutions are no more than 10 amino acid residues, such as 1,2,3,4,5,6,7,8,9, or 10 residues.

"Affinity" or "binding affinity" may be used to reflect the inherent binding capacity of interactions between members of a binding pair. The affinity of molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (K _D), which is the ratio of the dissociation rate constant and the binding rate constant (K _dis and K _on, respectively). Binding affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio affinity assay technique herein.

The terms "extracellular domain", "extracellular domain" or "ECD" are used interchangeably herein to refer to the region of a membrane protein that is outside the vacuolar membrane (e.g., the space outside the cell) when the full-length form of the membrane protein (e.g., transmembrane protein) is expressed from the cell. For the purposes herein, it is understood that references to ECD refer to the sequences and domains that make up the region, and that the ECD-containing protein is not required to be a membrane protein or that the domains are present outside the cell. For example, the soluble immunomodulatory protein may comprise the ECD sequence of a membrane protein fused to another moiety (e.g., a multimerization domain, such as an Fc region). The extracellular domain typically interacts with a specific ligand or a specific cell surface receptor, for example by specifically binding to a binding domain of the ligand or cell surface receptor. Examples of binding domains include cysteine-rich domains (CRDs). The extracellular domain of a TNFR superfamily member contains a TD domain (e.g., a CRD domain). Thus, references herein to the full length sequence of an ECD, including membrane proteins, and specific binding fragments thereof containing CRD or a portion thereof, in combination with a ligand.

Herein, an antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region, and may include native sequence Fc fragments and variant Fc fragments. The native sequence Fc fragment encompasses a variety of immunoglobulin Fc sequences that occur naturally, such as the Fc region of various Ig subtypes and their allotypes (GesturVidarsson et al ,IgG subclasses and allotypes:from structure to effector functions,20October 2014,doi:10.3389/fimmu.2014.00520.) in some embodiments, the Fc region is from the Fc region of an IgG, such as the Fc region of a human IgG in some embodiments, the Fc region is from the Fc region of an IgG1, igG2, igG3, or IgG4, such as the Fc region of a human IgG1, igG2, igG3, or IgG4 in some embodiments, the Fc region is the Fc region of a human IgG1, igG2, igG3, or IgG4 in one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 of the heavy chain or from Pro230 to the carboxy terminus in another embodiment, in other embodiments, the Fc fragment is a variant Fc fragment comprising a mutation, e.g., comprising an L234A-L235A mutation, unless otherwise indicated herein, the numbering of the amino acid residues in the Fc fragment is according to the EU numbering system, also known as the EU numbering system, e.g., kabat, E.A. et al, sequences ofProteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes ofHealth, bethesda, MD (1991), NIH Publication 91-3242.

The term "linker" as used herein refers to any molecule that enables direct ligation of different parts of a fusion protein. Examples of linkers that establish covalent linkages between different portions of the fusion protein include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol, polypropylene glycol. The term "peptide linker" according to the invention refers to a sequence of amino acids, wherein the sequence connects the amino acid sequence of a first part of a fusion protein to a second part of the fusion protein. For example, a peptide linker may link the TACI/BCMA portion of the fusion protein to an Fc domain or fragment thereof. For example, a peptide linker may also link the antibody to TACI/BCMA, such as linking the C-terminus of the antibody heavy chain to TACI/BCMA. Preferably, the peptide linker has a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide linker may or may not include predominantly the amino acid residues Gly, ser, ala or Thr. Useful linkers include glycine-serine polymers including, for example, (GSGGGGS) n (SEQ ID NO: 89), (GS) n (SEQ ID NO: 90), (GSGGS) n (SEQ ID NO: 91), (GGGGS) n (SEQ ID NO: 92) or (GGGS) n (SEQ ID NO: 93), where n is an integer of at least 1 (and for example 2,3,4, 5, 6,7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the linker of the invention is (GSGGGGS) n (SEQ ID NO: 89). Preferably, the linker of the invention is SEQ ID NO. 39.

The term "fusion" as used herein refers to a fusion formed by joining two or more initially separate proteins/genes/compounds. If the entity constituting the fusion is a protein, it is referred to as a fusion protein. Fusion proteins are encompassed within the scope of the fusion of the application. For example, linking TACI to Fc dimers may constitute TACI-Fc fusion proteins. The linkage between the two entity molecules constituting the fusion may or may not be achieved by a linker.

As used herein, the terms "first" and "second" are used in terms of Fc domains (Fc regions) or monomers, etc., to facilitate differentiation when there is more than one module of each class. The use of these terms is not intended to impart a particular order or orientation to the fusion protein unless explicitly stated as such.

The term "therapeutic agent" as described herein encompasses any substance that is effective in preventing or treating an immune disease or inflammation, such as an immune disease or inflammation, including, but not limited to, cytokines, hormones, cytotoxic or inhibitory agents (e.g., cytostatic agents affecting T cell and/or B cell proliferation), antibodies, or small molecule drugs or immunomodulators (e.g., immunosuppressants).

The term "effective amount" refers to an amount or dose of an antibody or fragment or composition or combination of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention. An "effective amount" may encompass a "therapeutically effective amount" or a "prophylactically effective amount".

"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are less than the therapeutically beneficial effects. The "therapeutically effective amount" preferably inhibits the measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100% relative to an untreated subject. "prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, since the prophylactic dose is administered in the subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cells.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to and facilitates detection of an agent (such as a polynucleotide probe or antibody) to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or in the case of enzymatic labels may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled.

The term "biological half-life" refers to the length of time it takes a substance (e.g., an immunomodulatory protein) to lose half its pharmacological or physiological activity or concentration. Biological half-life may be affected by elimination, excretion, degradation (e.g., enzymatic degradation/digestion) of a substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life may be assessed by determining the time it takes for the plasma concentration of a substance to reach half its steady state level ("plasma half-life").

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

The term "immune system disease or disorder" refers to any disease or disorder associated with an immune system abnormality, including but not limited to autoimmune or inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, and the like.

The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. for administration with the active substance.

The term "pharmaceutical composition" refers to a composition that exists in a form that is effective to allow the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered.

The term "pharmaceutical combination" or "combination product" refers to a non-fixed combination or fixed combination, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the chimeric or fusion proteins of the invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time constraints, or sequentially at the same or different time intervals, in separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and/or time intervals of the two or more active agents are preferably selected so that the combined use of the parts will produce an effect in the treatment of a disease or condition that is greater than that achieved by either component alone. The components can be in the form of separate preparations, and the preparations can be the same or different.

The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment (e.g., radiation therapy or surgery) to treat the diseases described herein. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, e.g., in a single capsule with a fixed ratio of active ingredients. Or such administration includes co-administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the pharmaceutical combination in treating the disorders or conditions described herein.

As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to administration of a drug prior to the occurrence of a sign or symptom of cancer, particularly in a subject at risk of cancer.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

"Subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, like a tissue sample or biopsy or puncture from a fresh, frozen and/or preserved organ or tissue, blood or any blood component, body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid, cells from a subject at any time during pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

TACI/BCMA chimeras and fusion proteins thereof

In some embodiments, the invention provides a TACI mutein that has more uniform charge on the surface of the protein, better stability, and better drug-forming properties.

In some embodiments, the TACI muteins of the present invention reduce the area of surface charge groups and hydrophobic groups after mutation, thereby reducing the risk of aggregation of TACI.

In some embodiments, the TACI muteins of the present invention are TACI mutated extracellular domains or functional fragments thereof, wherein a portion of the segment is replaced by a corresponding segment of BCMA. Thus, TACI muteins comprising such mutations are also referred to herein as TACI/BCMA chimeras.

In some embodiments, a TACI/BCMA chimera of the present invention comprises an extracellular domain of TACI or a functional fragment thereof wherein the N-terminal amino acid and/or C-terminal amino acid of the extracellular domain or functional fragment thereof is replaced with the N-terminal amino acid and/or C-terminal amino acid of the extracellular domain of BCMA.

In some embodiments, a TACI/BCMA chimera of the present invention comprises a TACI mutated extracellular domain or functional fragment thereof wherein the N-terminal amino acid of the extracellular domain or functional fragment thereof is replaced with the N-terminal amino acid of the extracellular domain of BCMA.

In some embodiments, the TACI/BCMA chimera of the present invention comprises a TACI mutated extracellular domain or functional fragment thereof wherein the N-terminal amino acid and the C-terminal amino acid of the extracellular domain or functional fragment thereof are replaced with the N-terminal amino acid and the C-terminal amino acid, respectively, of the extracellular domain of BCMA.

In some embodiments, the TACI/BCMA chimeras of the present invention further comprise a substitution to a patency risk site. In some embodiments, the pharmaceutical risk site is selected from positions 69, 72, 73, 74, 77, 78, 85, 102 and/or 103 of TACI. In some embodiments, the amino acid of the pharmaceutical risk site is mutated to a or D, e.g., D. In some embodiments, the substitution of the patency risk site is Y102D.

In some embodiments, the invention also provides fusion proteins comprising the TACI/BCMA chimeras of the invention. In a preferred embodiment, the TACI/BCMA chimeric of the invention is fused to another polypeptide, such as albumin, preferably an antibody Fc fragment, which may confer improved pharmacokinetic properties.

In some embodiments, the invention also provides fusion protein dimers formed by dimerization of two fusion protein chains of the invention.

The components of the TACI/BCMA chimeric or fusion proteins of the present invention are described in detail below. It will be appreciated by those skilled in the art that any combination of any of the features of these components is within the contemplation of the invention unless the context clearly indicates to the contrary. Moreover, it will be appreciated by those skilled in the art that unless the context clearly indicates to the contrary, the TACI/BCMA chimeric or fusion proteins thereof of the present invention may comprise any such combination of features.

II-1TACI ectodomains or functional fragments thereof

TACI is a member of the tumor necrosis factor receptor family characterized by having an extracellular domain (ECD) comprising a cysteine-rich pseudo-repeat domain (CRD). TACI is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudorepeats (CRD 1 and CRD 2), a transmembrane domain, and a cytoplasmic domain that interacts with CAMLs (calcium modulator and cyclophilin ligand), integral membrane proteins located AT intracellular vesicles, which are co-inducers of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with a subset of B cells and T cells. The TACI receptor binds to two members of the Tumor Necrosis Factor (TNF) ligand family. One ligand is named BAFF (B-cell activator of TNF family). Another ligand has been named APRIL. Both ligands are also bound by B cell maturation receptors (BCMA). Binding of TACI receptors to their ligands BAFF or APRIL stimulates B cell responses, including T cell independent B cell antibody responses, isotype switching, and B cell homeostasis. Binding of BAFF or APRIL stimulates B cell responses including T cell independent B cell antibody responses, isotype switching and B cell homeostasis.

In some embodiments, the TACI is human TACI. In some embodiments, the human TACI comprises or consists of the amino acid sequence set forth in SEQ ID No. 1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the full length sequence of human TACI comprises or consists of the amino acid sequence shown in SEQ ID NO. 1. In some embodiments, the human TACI protein is a type III membrane protein and lacks a signal peptide, and the N-terminal methionine is removed after expression in eukaryotic cells. In some embodiments, the mature TACI protein does not contain an N-terminal methionine as shown in SEQ ID NO. 1.

The structure and characterization of wild-type human TACI can be found in Uitprot O14836 (https:// www.uniprot.org/uniprotkb/O14836/entry). Human TACI typically comprises 3 domains, the extracellular domain, corresponding to amino acids 1-165, the transmembrane region, corresponding to amino acids 166-186, and the cytoplasmic domain, corresponding to amino acids 187-293.

The extracellular domain of TACI (e.g., amino acid residues 1-165 of SEQ ID NO: 1; ECD shown in SEQ ID NO: 29) contains two cysteine-rich domains (CRDs), each exhibiting affinity for binding to BAFF and APRIL. The first cysteine-rich domain (CRD 1) corresponds to or contains amino acid residues 34-66 of the sequence shown in SEQ ID NO. 1. The second cysteine-rich domain (CRD 2) corresponds to or contains amino acids 71-104 of the sequence shown in SEQ ID NO. 1. TACI also contains in the extracellular domain a stem region of about 60 amino acids after the second cysteine repetitive sequence, corresponding to or containing amino acid residues 105-165 of the sequence shown in SEQ ID NO. 1.

In some embodiments, the extracellular domain of TACI comprises or consists of the amino acid sequence at positions 1-165 of SEQ ID No. 1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the extracellular domain of TACI comprises or consists of an amino acid sequence shown as SEQ ID NO. 29, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, CRD2 comprises or consists of the amino acid sequence set forth in SEQ ID NO. 58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the functional fragment of TACI ECD comprises an amino acid sequence that binds to APRIL, BAFF, or APRIL/BAFF heterotrimers.

In some embodiments, the functional fragment of TACI ECD lacks a portion of the N-terminal residue of ECD, which residue corresponds to 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66 or 67 consecutive amino acid deletions at the N-terminus of the ECD sequence shown in SEQ ID NO. 1.

In some embodiments, a functional fragment of TACI ECD lacks a partial stem region of ECD, e.g., lacks 55、54、53、52、51、50、49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2 or 1 contiguous amino acids at the C-terminus.

In some embodiments, the functional fragment of TACI ECD comprises CRD2, or only comprises intact CRD2. In some embodiments, the functional fragment of TACI ECD consists of CRD2.

In some embodiments, the functional fragment of TACI ECD comprises CRD2 and a partial stem region. In some embodiments, the functional fragment of TACI ECD further comprises a sequence between CRD1 and CRD2, e.g., corresponding to any amino acid sequence between positions 68-70 of SEQ ID NO. 1. In some embodiments, the functional fragment of TACI ECD does not comprise CRD1 or any fragment thereof.

In some embodiments, the functional fragment of TACI ECD contains a partial stem region, e.g., comprising a sequence of TACI corresponding to SEQ ID NO:1, and the following fragment of the amino acid sequence shown in SEQ ID NO: amino acid residues 105, 105 to 106, 105 to 107, 105 to 108, 105 to 109, 105 to 110, 105 to 111, 105 to 112, 105 to 113, 105 to 114, 105 to 115, 105 to 116, 105 to 117, 105 to 118, 105 to 119, 105 to 120, 105 to 121, 105 to 122, 105 to 123, 105 to 124, 105 to 125, 105 to 126, 105 to 127, 105 to 128, 105 to 129, and amino acid residues 105 to 130, amino acid residues 105 to 131, amino acid residues 105 to 132, amino acid residues 105 to 133, amino acid residues 105 to 134, amino acid residues 105 to 135, amino acid residues 105 to 136, amino acid residues 105 to 137, amino acid residues 105 to 138, amino acid residues 105 to 139, amino acid residues 105 to 140, amino acid residues 105 to 141, amino acid residues 105 to 142, amino acid residues 105 to 143, amino acid residues 105 to 144, amino acid residues 105 to 145, amino acid residues 105 to 146, amino acid residues 105 to 147, amino acid residues 105 to 148, amino acid residues 105 to 149, amino acid residues 105 to 150, amino acid residues 105 to 151, amino acid residues 105 to 152, amino acid residues 105 to 153, and amino acid residues 105 to 154.

In some embodiments, the functional fragment of TACI ECD comprises a fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO. 1, amino acid residues 67 to 118, amino acid residues 67 to 117, amino acid residues 67 to 116, amino acid residues 67 to 115, amino acid residues 67 to 114, amino acid residues 67 to 113, amino acid residues 67 to 112, amino acid residues 67 to 111, amino acid residues 67 to 110, amino acid residues 67 to 109, amino acid residues 67 to 108, amino acid residues 67 to 107, Amino acid residues 67 to 106, 67 to 105 or 67 to 104, amino acid residues 68 to 118, 68 to 117, 68 to 116, 68 to 115, 68 to 114, 68 to 113, 68 to 112, 68 to 111, 68 to 110, 68 to 109, 68 to 108, 68 to 107, 68 to 106, 68 to 105 or 68 to 104, 69 to 118, Amino acid residues 69 to 117, 69 to 116, 69 to 115, 69 to 114, 69 to 113, 69 to 112, 69 to 111, 69 to 110, 69 to 109, 69 to 108, 69 to 107, 69 to 106, 69 to 105 or 69 to 104, 70 to 118, 70 to 117, 70 to 116, Amino acid residues 70 to 115, 70 to 114, 70 to 113, 70 to 112, 70 to 111, 70 to 110, 70 to 109, 70 to 108, 70 to 107, 70 to 106, 70 to 105 or 70 to 104, 71 to 118, 71 to 117, 71 to 116, 71 to 115, 71 to 114, Amino acid residues 71 to 113, amino acid residues 71 to 112, amino acid residues 71 to 111, amino acid residues 71 to 110, amino acid residues 71 to 109, amino acid residues 71 to 108, amino acid residues 71 to 107, amino acid residues 71 to 106, amino acid residues 71 to 105 or amino acid residues 71 to 104. In some embodiments, the functional fragment of a TACI ECD comprises or consists of amino acids 13-118, or 68-110 of the ECD.

In some embodiments, the functional fragment of a TACI ECD comprises or consists of the amino acid sequence set forth in SEQ ID No. 30, 31 or 58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence shown in SEQ ID NO. 31.

In some embodiments of the invention TACI ECD or functional fragment thereof also encompasses ECD variants with mutations or functional fragments thereof. In some embodiments, the functional fragment of TACI ECD comprises K77E, F Y and/or Y102D.

In some embodiments, the functional fragment of TACI ECD comprises Y102D. In some embodiments, the functional fragment of a TACI ECD comprises or consists of the amino acid sequence set forth in SEQ ID No. 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the functional fragment of TACI ECD comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO. 33 and has Y102D. In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence shown in SEQ ID NO. 33.

In some embodiments, the functional fragment of TACI ECD comprises K77E, F Y and Y102D. In some embodiments, the functional fragment of a TACI ECD comprises or consists of the amino acid sequence shown in SEQ ID NO. 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the functional fragment of TACI ECD comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO. 32 and has K77E, F Y and Y102D. In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence shown in SEQ ID NO. 32.

II-2TACI/BCMA chimeras

The invention discovers that the protein structure (6-42, PDB ID:1XU 2) of the BCMA of the TACI same family protein has high homology with the structure of the TACI, is basically consistent with the binding site of the ligand, and meanwhile, the N end part and the C end part of the TACI are not involved in the binding of the ligand (figure 1), more importantly, the analysis result of Discovery studio software shows that the BCMA has balanced surface charge distribution and aggregation risk which is obviously lower than that of the TACI.

Therefore, a series of chimeric molecules of BCMA and TACI extracellular domains are designed, and the N-terminal amino acid or the C-terminal amino acid of the TACI is replaced by the corresponding sequence of the BCMA on the basis of keeping the binding sites of the TACI and the ligand, or simultaneously, the N-terminal amino acid and the C-terminal amino acid of the TACI are replaced by the corresponding sequence of the BCMA, and optionally, partial drug-forming risk sites are subjected to amino acid replacement, so that the areas of surface charge groups and hydrophobic groups of the TACI protein are reduced, and the aggregation risk of the TACI is reduced.

BCMA is a member of the tumor necrosis factor receptor family characterized by having an extracellular domain (ECD) that contains a cysteine-rich pseudo-repeat domain (CRD). BCMA is a membrane-bound receptor having an extracellular domain containing a single CRD, a transmembrane domain, and a cytoplasmic domain containing a TRAF binding site for binding to a TRAF signaling molecule. BCMA binds to cognate ligands APRIL and BAFF, but with weaker affinity to BAFF. The binding of BCMA to BAFF is reported to be two to three orders of magnitude weaker than that between BAFF and other cognate receptors BAFF-R and TACI.

In some embodiments, the BCMA is human BCMA. In some embodiments, human BCMA comprises or consists of the amino acid sequence shown in SEQ ID No. 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the full length human BCMA sequence comprises the amino acid sequence shown in SEQ ID NO. 2. In some embodiments, the BCMA protein is a type II membrane protein and lacks a signal peptide, and the N-terminal methionine is removed after expression in eukaryotic cells. In some embodiments, the mature BCMA protein does not contain an N-terminal methionine as shown in SEQ ID NO. 2.

The structure and characterization of wild-type human BCMA can be found in Uitprot Q02223 (https:// www.uniprot.org/uniprotkb/Q02223/entry). Human BCMA typically comprises 3 domains, the extracellular domain, corresponding to amino acids 1-54, the transmembrane region corresponding to amino acids 55-77, and the cytoplasmic domain corresponding to amino acids 78-184. The extracellular domain of BCMA contains a cysteine-rich domain (CRD) that exhibits affinity for binding to APRIL and to a lesser extent to BAFF. In some embodiments, the CRD comprises amino acid residues 7-41 of the sequence set forth in SEQ ID NO. 2.

In some embodiments, the extracellular domain of BCMA comprises or consists of the amino acid sequence of SEQ ID No. 2 at positions 1-54, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the extracellular domain of BCMA comprises or consists of the amino acid sequence shown in SEQ ID No. 57 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the N-terminus and/or C-terminus of a TACI ECD or functional fragment thereof (e.g., CRD 2-only ECD, e.g., TACI (68-110)) is replaced with the corresponding sequence of BCMA.

In some embodiments, the N-terminus of TACI ECD or a functional fragment thereof refers to the N-terminal amino acid of TACI before the amino acid at position Y79 of SEQ ID NO. 1.

In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminus amino acid of TACI after amino acid 99, amino acid 100, amino acid 101, amino acid 102, amino acid 103, amino acid 104, amino acid 105, amino acid 106, amino acid 107, amino acid 108, amino acid 109, or amino acid 110 of SEQ ID NO. 1.

In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid of TACI corresponding to amino acid 99 of SEQ ID NO. 1. In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid after amino acid 105 corresponding to SEQ ID NO. 1.

In some embodiments, the corresponding N-terminal amino acid sequence of BCMA that may be used to replace the N-terminal of TACI ECD or a functional fragment thereof is selected from the amino acid sequences of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, or 7-13 of SEQ ID NO. 2.

In some embodiments, the corresponding N-terminal amino acid sequence of BCMA for substitution comprises, or consists of, the amino acid sequence of any one of SEQ ID NOs 40-46.

In some embodiments, the corresponding C-terminal amino acid sequence of BCMA that may be used to replace the C-terminal end of TACI ECD or a functional fragment thereof is selected from the group consisting of the amino acid sequences of BCMA corresponding to positions 37-47, 37-46, 37-45, 37-44 of SEQ ID NO. 2, or the amino acids corresponding to positions 43-44 or 43-46 of SEQ ID NO. 2.

In some embodiments, the corresponding N-terminal amino acid or C-terminal amino acid sequence of BCMA for substitution may comprise mutations, e.g., substitutions, for improving binding affinity, increasing stability, and/or improving drug formation. In some embodiments, the C-terminal amino acid sequence of BCMA for substitution comprises a mutation at position 39 and/or 42.

In some embodiments, the C-terminal amino acid sequence of BCMA for substitution comprises N42A. In some embodiments, the C-terminal amino acid sequence of BCMA for substitution comprises N42Q. In some embodiments, the C-terminal amino acid sequence of BCMA for substitution comprises R39D. In some embodiments, the C-terminal amino acid sequence of BCMA for substitution comprises N42A-R39D.

In some embodiments, the corresponding C-terminal amino acid sequence of BCMA for substitution comprises, or consists of, the amino acid sequence set forth in any one of SEQ ID NOs 47-55.

In some embodiments, the number of amino acids of the N-terminal or C-terminal amino acid sequence of BCMA used for substitution is equal to or within 1-3 amino acids of the number of amino acids of the N-terminal or C-terminal amino acid sequence of the ECD or functional fragment thereof of the TACI being substituted.

Thus, in some embodiments, the TACI/BCMA chimeras of the present invention comprise the following structure:

BCMAN terminal amino acid-TACI moiety, wherein the TACI moiety is an ECD or functional fragment thereof lacking the N-terminus, or

BCMAN terminal amino acid-TACI moiety-BCMAC terminal amino acid, wherein the TACI moiety is an ECD or functional fragment thereof lacking an N-terminus and a C-terminus.

Thus, herein, "BCMAN terminal amino acid-TACI moiety" refers to a polypeptide obtained after substitution of the N-terminal amino acid of TACI with the N-terminal amino acid of BCMA. "BCMAN terminal amino acid-TACI part-BCMAC terminal amino acid" refers to a polypeptide obtained after substitution of the N-terminal amino acid of TACI with the N-terminal amino acid of BCMA and substitution of the C-terminal amino acid of TACI with the C-terminal amino acid of BCMA.

In some embodiments, TACI portions suitable for use in TACI/BCMA chimeras of the present invention may comprise mutations, e.g., substitutions.

In some embodiments, the TACI portion of a TACI/BCMA chimera suitable for use in the present invention further comprises a mutation at a pharmaceutical risk site, e.g., an amino acid site at risk of aggregation, e.g., an amino acid that replaces one or several of the pharmaceutical risk sites of the TACI portion. In some embodiments, if the TACI moiety comprises a pharmaceutical risk site, it is replaced with an amino acid that reduces that risk. In some embodiments, if the TACI moiety comprises an amino acid that is at risk of aggregation, it is replaced with an amino acid that reduces the risk of aggregation. In some embodiments, the pharmaceutical risk site is selected from amino acids 69, 72, 73, 74, 77, 78, 85, 102 or 103 corresponding to SEQ ID NO. 1. For example, the amino acid of the patency risk site is mutated to a or D.

In some embodiments, the TACI moiety comprises a mutation at position Y102, e.g., a substitution, e.g., Y102D.

In some embodiments, the TACI portion comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs 31-38, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity thereto, or an amino acid sequence having 1,2, 3, 4 or 5 amino acid changes (e.g., substitutions, deletions or additions, e.g., conservative substitutions; or substitutions, e.g., at a pharmaceutical risk site) thereto.

In some embodiments, the TACI portion consists of the amino acid sequence shown in any one of SEQ ID NOs 31-38.

In some embodiments, the BCMA portion (e.g., at the BCMAN terminus or the BCMAC terminus, preferably the BCMAC terminus) of the TACI/BCMA chimera suitable for use in the present invention further comprises a mutation, e.g., a substitution, e.g., a mutation at a pharmaceutical risk site, e.g., a mutation resulting in an N-glycosylation site, e.g., an amino acid that replaces one or several of the pharmaceutical risk sites of the BCMA portion, for improving binding affinity, increasing stability and/or improving drug formation. In some embodiments, if the BCMA moiety comprises a pharmaceutical risk site, it is replaced with an amino acid that reduces that risk. In some embodiments, if the BCMA moiety comprises an amino acid having glycosylation (e.g., N-glycosylation), it is replaced with another amino acid. In some embodiments, the pharmaceutical risk site is the amino acid corresponding to position 39 or 42 of SEQ ID NO. 2. For example, the amino acid of the patency risk site is mutated to D, A or Q.

In some embodiments, the BCMAN terminus comprises, or consists of, an amino acid sequence set forth in any one of SEQ ID NOs 40-46, or an amino acid sequence having at least 80% or 90% identity thereto, or an amino acid sequence having 1,2, or 3 amino acid changes (e.g., substitutions, deletions, or additions, such as conservative substitutions, or substitutions, such as at a site of risk of patency) thereto.

In some embodiments, BCMAN consists of the amino acid sequence set forth in any one of SEQ ID NOS.40-46.

In some embodiments, the BCMAC terminus comprises a mutation, e.g., a substitution, at position R39, e.g., R39D. In some embodiments, the BCMAC terminus comprises a mutation at position N42, e.g., a substitution, e.g., N42A or N42Q. In some embodiments, the BCMAC terminus comprises a mutation at position R39 and a mutation at position N42, e.g., R39D-N42A.

In some embodiments, the BCMAC terminus comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs 47-55, or an amino acid sequence having at least 80% or 90% identity thereto, or an amino acid sequence having 1 or 2 amino acid changes (e.g., substitutions, deletions or additions, such as conservative substitutions or substitutions, such as at a site of risk of pharmacy) thereto.

In some embodiments, the BCMAC terminus consists of the amino acid sequence set forth in any one of SEQ ID NOS.47-55.

The TACI/BCMA chimera of the present invention comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs 59-80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity thereto.

II-3 TACI/BCMA chimeric fusion proteins

In one aspect, the invention also provides a fusion protein comprising the TACI/BCMA chimera of the invention. In a preferred embodiment, the TACI/BCMA chimera of the invention is fused to another polypeptide, such as albumin, more preferably an antibody Fc fragment, which may confer improved pharmacokinetic properties. In some embodiments, the half-life of the TACI/BCMA chimeric-Fc fusion protein can be extended by FcRn-mediated recovery of in vivo circulation.

In one embodiment, the invention provides a TACI/BCMA chimeric fusion protein comprising a TACI/BCMA chimeric of the invention fused to an Fc region, preferably the Fc region is fused at the C-terminus of the TACI/BCMA chimeric of the invention, e.g., the N-terminus of the Fc region is fused to the C-terminus of the chimeric.

In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In some embodiments, the Fc region comprises a complete hinge region at its N-terminus, i.e., it starts at position E216 to the C-terminus corresponding to the human IgG1 heavy chain constant region. In some embodiments, the Fc region has a mutation at its N-terminal hinge region corresponding to cysteine at position 220 of IgG1 to serine, i.e., comprising a 220S mutation.

The Fc region may also lack a portion of the hinge region, e.g., starting at position D221 to the C-terminus, corresponding to the human IgG1 heavy chain constant region.

In one embodiment, the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO. 81 or 82 or an amino acid sequence having at least 90% identity thereto, e.g.95%, 96%, 97%, 98%, 99% or more.

In one embodiment, the Fc region of a fusion protein suitable for use in the present invention lacks lysine K (K447 del) at the C-terminus.

In one embodiment, the Fc region comprises or consists of the amino acid sequence shown as SEQ ID NO 83 or 84 or an amino acid sequence having at least 90% identity thereto, e.g.95%, 96%, 97%, 98%, 99% or more identity thereto (optionally also lacking a C-terminal lysine).

The binding molecules of the invention, such as the Fc region in antibodies, may also be mutated to obtain the desired properties. Mutations to the Fc region are known in the art.

In one embodiment, the Fc region is modified in the nature of its effector function (e.g., complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type isotype Fc region. In one embodiment, effector function is reduced or eliminated by a method selected from the group consisting of using an Fc isoform naturally having reduced or eliminated effector function, and Fc region modification. In a preferred embodiment, the Fc region has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions, e.g., comprising mutations that effect the above functions.

In one embodiment, modifications in the Fc region that alter the binding affinity for one or more Fc receptors may also be included. In one embodiment, the Fc receptor is an fcγ receptor, in particular a human fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to an fcγ receptor. In a further preferred embodiment, the Fc region may have mutations that result in increased serum half-life, e.g. mutations that improve the binding of the Fc fragment to FcRn.

For example, in some embodiments, the Fc region for use in the invention has reduced binding to fcγ receptors and the Fc fragment for use in the invention has reduced binding to fcγ receptors of L234A/L235A mutation, L234A/L235E mutation, G237A mutation, or L234A/L235E/G237A. For example, in some embodiments, the Fc region used in the invention has mutations that improve binding of the Fc fragment to FcRn, such as YTE mutations (M252Y/S254T/T256E) or LS mutations (M428L/N434S) that enhance binding of the Fc fragment to FcRn. Exemplary mutations are described, for example, in WO2002060919A2, which is incorporated herein by reference in its entirety.

In some embodiments, the Fc region comprises both the mutation that reduces binding to fcγ receptor and the mutation that improves binding of the Fc fragment to FcRn.

In some embodiments, the Fc region comprises L234A/L235E/G237A and M252Y/S254T/T256E, and optionally a C-terminal deleted lysine.

In some embodiments, the Fc region comprises L234A/L235E/G237A and M428L/N434S, and optionally a C-terminal deleted lysine.

In one embodiment, the Fc region comprises or consists of an amino acid sequence shown in any of SEQ ID NOs 85-88 and 100-103 or an amino acid sequence having at least 90% identity thereto, e.g., 95%,96%,97%,98%,99% or more identity.

In one embodiment, the Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 85 or 87 and comprising the mutation L234A/L235E/G237A.

In one embodiment, the Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO 86 or 88 and comprising the mutations L234A/L235E/G237A and C-terminally deleted lysines.

In one embodiment, the Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 100 or 102 and comprising the mutations L234A/L235E/G237A and M252Y/S254T/T256E.

In one embodiment, the Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 101 or 103 and comprising the mutations L234A/L235E/G237A, M252Y/S254T/T256E and C-terminal deleted lysine.

In some embodiments, the TACI/BCMA chimera is fused to Fc directly or through a linker, e.g., the C-terminus of the chimera is fused to the N-terminus of Fc directly or through a linker. As will be apparent to those of skill in the art, suitable linkers for linking the TACI/BCMA and Fc regions in the fusion proteins and dimer molecules of the present invention may be any linker known in the art. In some embodiments, the linker may comprise an IgG1 hinge, or may comprise a linker sequence ：(GSGGGGS)_n(SEQ ID NO:94)、(GS)_n(SEQ ID NO:95)、(GSGGS)_n(SEQ ID NO:96)、(GGGGS)_n(SEQ ID NO:97) or (GGGS) _n (SEQ ID NO: 98) selected from the group consisting of, wherein n is an integer of at least 1, such as 1, 2,3, 4, or 5.

In one embodiment, the linker is (GSGGGGS) _n (SEQ ID NO: 99), where n=1-3. In one embodiment, the linker comprises or is the amino acid sequence shown in SEQ ID NO. 39.

In a further aspect, the invention also provides a dimer molecule comprising a TACI/BCMA chimera of the invention fused to an Fc region.

In some embodiments, the invention provides a TACI/BCMA chimeric-Fc dimer protein wherein the first monomer and the second monomer comprise or consist of i) a TACI/BCMA chimeric, ii) a linker (optionally present or absent), and iii) an Fc region, respectively, from the N-terminus to the C-terminus. In some embodiments, the dimeric protein is a homodimer. In some embodiments, the monomers have the same amino acid sequence. In some embodiments the TACI/BCMA chimeric-Fc protein dimers of the invention comprise two identical monomers.

In some embodiments, the TACI/BCMA chimeric-Fc fusion proteins of the invention or monomers thereof

(Ii) Comprising or consisting of the amino acid sequence of any one of SEQ ID NOs 5 to 28, or

(Iii) Comprising an amino acid change of 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) compared to the amino acid sequence of any one of SEQ ID NOs 5 to 28.

In some embodiments, the TACI/BCMA chimeric-Fc proteins of the invention, or the protein dimer monomers of the invention, consist of the amino acid sequence of any one of SEQ ID NOs 5-28.

III polynucleotides, vectors and hosts

The present invention provides nucleic acids encoding any of the above TACI/BCMA chimeras or any of the fusion proteins or fusion protein dimers thereof or any of the monomers or domains. The polynucleotide sequence encoding the mutein of the present invention can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of the existing sequence encoding the wild-type TACI using methods well known in the art. In addition, polynucleotides and nucleic acids of the invention may comprise a segment encoding a secretion signal peptide and are operably linked to a segment encoding a mutein of the invention, thereby allowing for the guidance of secretory expression of the mutein of the invention.

The invention also provides vectors comprising the nucleic acids of the invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). In a preferred embodiment, the expression vector of the invention is a pCDNA expression vector, such as a pCDNA3.1 expression vector.

The invention also provides a host cell comprising said nucleic acid or said vector. Host cells suitable for replication and supporting expression of TACI/BCMA chimeras or fusion proteins or fusion protein dimers are well known in the art.

Such cells can be transfected or transduced with a specific expression vector and a large number of vector-containing cells can be grown for inoculation of a large scale fermenter to obtain a sufficient amount of TACI/BCMA chimera or fusion protein dimer thereof for clinical use.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., CHO cell or 293 cell).

Examples of mammalian host cell lines that can be used are monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line (293 or 293T cells or HEK293 cells), baby mouse kidney cells (BHK), mouse sertoli (TM 4 cells), monkey kidney cells (CV 1), african green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG 2), mouse mammary tumor cells (MMT 060562), TRI cells, MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr-CHO cells, and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.

In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, HEK293 cell, human Embryonic Kidney (HEK) cell or lymphocyte (e.g., Y0, NS0, sp20 cell).

IV preparation method

In a further aspect, the invention provides a method of preparing a TACI/BCMA chimera of the invention or a fusion protein or fusion protein dimer thereof, wherein said method comprises culturing a host cell comprising a nucleic acid encoding said TACI/BCMA chimera or fusion protein dimer thereof, as provided above, under conditions suitable for expression of said TACI/BCMA chimera or fusion protein dimer thereof, and optionally recovering said TACI/BCMA chimera or fusion protein dimer thereof from said host cell (or host cell culture medium).

In one embodiment, a vector comprising a nucleic acid encoding a TACI/BCMA chimera or fusion protein thereof or fusion protein dimer is transferred into cells for expression, followed by harvesting the cells (or cell culture supernatant), extracting the TACI/BCMA chimera or fusion protein thereof or fusion protein dimer, and purifying to obtain the TACI/BCMA chimera or fusion protein thereof or fusion protein dimer.

In a specific embodiment, the purification method is an affinity purification method. In another specific embodiment, the purification method is ion exchange purification. In some embodiments, the purification is by filtration using a gel filtration chromatography column.

V. properties of the chimeric TACI/BCMA or fusion proteins or fusion protein dimers of the invention

The chimeric or fusion protein dimer thereof provided herein of TACI/BCMA can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art.

The protein surface charge of the chimeric of TACI/BCMA obtained by the invention is more uniform, so that the chimeric has better stability and better drug property.

The chimeric TACI/BCMA and fusion proteins thereof obtained by the invention have better stability (e.g. thermal stability) and pharmacokinetic data and have longer half-life compared with the TACI proteins and fusion proteins thereof known in the prior art. The chimeric fusion protein of TACI/BCMA obtained by the invention has better stability under the acceleration condition compared with the TACI fusion protein or the variant thereof known in the prior art, wherein the thermal stability of part of molecules is obviously better than ALPN.

In some embodiments, the chimeric fusion protein of TACI/BCMA obtained according to the present invention has one or more properties selected from the group consisting of:

a) The expression quantity in the cells is high, and the purification is easy;

b) Specifically binds BAFF and APRIL, for example, by biological optical interferometry (ForteBio) assay, and has a K _D of less than or equal to about 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM or 0.4nM, or greater than or equal to about 0.1nM or 0.2nM, or between any of the values, and has a K _D of less than or equal to about 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM or 0.4nM, or greater than or equal to about 0.1nM or 0.2nM, or between any of the values;

c) Effective to inhibit binding of BAFF to IM-9 cells, e.g., with an IC50 of less than or equal to about 5nM, 4nM, 3nM, 2nM or 1.5nM, and effective to inhibit binding of APRIL to IM-9 cells, e.g., with an IC50 of less than or equal to about 5nM, 4nM, 3nM, 2nM 1.5nM, 1nM, 0.9nM,

0.8NM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM or 0.2nM;

d) Effective to inhibit BAFF/APRIL-induced surface expression of (human) TACI, e.g., having an IC50 of less than or equal to about 16nM, 15nM, 14nM, 13nM, 12nM, as determined by TACI/NF- κ B ReporterJurkat signaling,

11NM, 10nM, 9nM, 8nM, 7nM, 6nM or 5.5nM;

e) Effective to inhibit BAFF/APRIL-induced surface expression of (human) BCMA, e.g., an IC50 of less than or equal to about 16nM, 15nM, 14nM, 13nM, as determined by BCMA/NF- κ BReporter Jurkat signaling,

12NM, 11nM, 10nM, 9nM, 8nM, 7nM, 6Nm, 5.5nM, 5nM, 4.5nM or 4nM;

f) Effective to inhibit BAFF/APRIL-induced spleen cell proliferation, e.g., an IC50 of less than or equal to about 4.5nM, 4nM,

3.5NM, 3nM, 2.5nM, 2nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM or 0.6nM;

g) Effectively inhibit B cell proliferation induced by BAFF/APRIL;

h) The heat stability is better;

i) The medicine has better medicine property;

j) Has better acceleration stability, such as better than known molecules RC-18 and/or ALPN-303;

k) Effectively inhibit BAFF-induced proliferation of spleen cells and/or B cells and/or different B cell subsets in the spleen;

l) is effective in inhibiting proliferation of spleen cells and/or B cells and/or different B cell subsets in the spleen, e.g. in KLH immune models, e.g. better than the known molecule RC-18;

m) is effective to reduce BAFF-induced IgA, igM and/or IgG levels in serum;

n) inhibits proliferation of MZ cells, GC B cells, plasma cells more effectively, e.g. better than known molecules ALPN-303;

o) has better pharmacokinetics, e.g. better than known molecules RC-18 and/or ALPN-303;

p) has a longer half-life (e.g., plasma half-life), e.g., a half-life (e.g., plasma half-life) of greater than or equal to about 45 hours, e.g., greater than or equal to about 46, 47, 48, 49, or 50 hours, and/or

Q) has a lower clearance, for example a clearance of less than about 1.8, 1.7, 1.6 or 1.5mL/kg/h.

VI pharmaceutical composition and pharmaceutical preparation

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising TACI/BCMA chimeras or fusion proteins or fusion protein dimers thereof or compositions comprising polynucleotides encoding TACI/BCMA chimeras or fusion proteins or fusion protein dimers thereof. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients as known in the art, including buffers.

Pharmaceutical compositions comprising the TACI/BCMA chimeric or fusion proteins or fusion protein dimers thereof of the invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Suitable formulations depend on the chosen route of administration.

TACI/BCMA chimeras or fusion proteins or fusion protein dimers may be formulated in free acid or base, neutral or salt form into compositions. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts (acid addition salt), such as those formed with the free amino groups of the proteinaceous composition, or with inorganic acids such as, for example, hydrochloric or phosphoric acid, or with organic acids such as acetic, oxalic, tartaric or mandelic acid.

VII combination of

In one aspect, the invention also provides a pharmaceutical combination or combination product comprising a TACI/BCMA chimera of the invention or a fusion protein or fusion protein dimer thereof, and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents affecting T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)). The pharmaceutical combinations or combination products of the invention may be used in the methods of treatment of the invention.

In some embodiments, the pharmaceutical combination or combination product is for use in preventing or treating a B cell related disease or immune system disease or inflammation.

In some embodiments, the pharmaceutical combination or combination product is for use in the manufacture of a medicament for the prevention or treatment of a B cell related disease or immune system disease or inflammation.

Therapeutic methods and uses

In one aspect, the invention relates to a method of preventing or treating a disease, such as a B cell or autoantibody related disease or an immune system disease (e.g., autoimmune disease) or inflammation in a subject, the method comprising administering to the subject an effective amount of any TACI/BCMA chimera described herein or fusion protein dimer or pharmaceutical composition or pharmaceutical combination thereof. In some embodiments, the disease is a disease associated with abnormal expression (increased concentration) or activity of BAFF and/or APRIL or BAFF/APRIL heterotrimers, e.g., as compared to a sample (e.g., blood or serum) of a healthy individual.

In some embodiments, the B cell or autoantibody related disease or immune system disease is a B cell or autoantibody mediated disease, e.g., a B cell or autoantibody mediated autoimmune disease. In some embodiments, the B cell mediated disease is a B cell expansion or abnormal activation related disease, such as a B cell expansion or abnormal activation autoimmune disease. In some embodiments, the autoantibody mediated disease refers to unwanted autoantibody production due to abnormal proliferation or activation of B cells, or abnormal autoantibody production.

In some embodiments, the disease associated with B cell expansion is a disease of abnormal B cell proliferation (e.g., an autoimmune disease), such as compared to a sample of a healthy individual (e.g., blood or serum). In some embodiments, the B cell or autoantibody mediated disease results in overproduction of autoantibodies against autoantigens or in abnormal autoantibody production due to abnormal B cell activation.

In one aspect, the invention relates to a method of inhibiting B cell proliferation in an individual, the method comprising administering to the subject an effective amount of any TACI/BCMA chimeric or fusion protein dimer thereof described herein.

In some embodiments, treatment of the disease will benefit from inhibiting BAFF and/or APRIL or BAFF/APRIL heterotrimer-related signaling pathways. In some embodiments, the treatment of the disease will benefit from inhibiting B cell proliferation or lymphocyte proliferation or inhibiting autoantibodies.

In some embodiments, the autoimmune disease includes, but is not limited to, lupus such as systemic lupus erythematosus, rheumatoid arthritis, igA nephropathy (IgAN) or membranous nephropathy, chronic kidney disease such as Sjogren's syndrome, myasthenia gravis, idiopathic Thrombocytopenic Purpura (ITP), warm-blooded autoimmune hemolytic anemia (wAIHA), multiple Sclerosis (MS), coronary heart disease (CAD), or thyroid eye disease.

The TACI/BCMA chimeric or fusion protein dimer thereof of the present invention (and pharmaceutical compositions comprising the same, optionally additional therapeutic agents) may be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending in part on whether the administration is short-term or long-term, the administration may be by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single dosing or multiple dosing at multiple time points, bolus dosing, and pulse infusion.

For the prevention or treatment of a disease, the appropriate dosage of the TACI/BCMA chimeric or fusion protein dimer thereof of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in one treatment or over a series of treatments.

In a further aspect, the invention also provides the use of a TACI/BCMA chimera of the invention or a fusion protein or fusion protein dimer thereof or a pharmaceutical composition or pharmaceutical combination thereof for the manufacture of a medicament for use in the aforementioned method, e.g. for the treatment of a disease associated with said B cells or autoantibodies or a disease of the immune system, e.g. an autoimmune disease or inflammation.

In a further aspect, the invention also provides a TACI/BCMA chimera according to the invention or a fusion protein dimer or a pharmaceutical composition or a pharmaceutical combination thereof for use in therapy, e.g. in the aforementioned methods (e.g. for the treatment of a disease associated with said B cells or autoantibodies or a disease of the immune system (e.g. autoimmune disease) or inflammation) or for use.

In a further aspect, the invention also provides the use of a TACI/BCMA chimera of the invention or a fusion protein dimer or a pharmaceutical composition or a pharmaceutical combination thereof for the aforementioned method, e.g. for the treatment of a disease associated with said B cells or autoantibodies or a disease of the immune system (e.g. autoimmune disease) or inflammation. The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed in any way as, limiting the scope of the invention.

These and other aspects and embodiments of the application are described in and exemplified by the following examples in the figures (which are briefly described to follow) and in the following detailed description. Any or all of the features discussed above and throughout the present application may be combined in various embodiments of the application. The following examples further illustrate the application, however, it is to be understood that the examples are presented by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

EXAMPLE 1 TACI analysis and modification

Full-length TACI comprises 293 amino acid residues, and protease cleavage sites are present at multiple positions of the extracellular domain, resulting in a very easy cleavage after expression of extracellular full-length TACI, whereas two Cysteine-rich domains (CRDs) are reported to be present in the TACI extracellular domain, CRD1 and CRD2, respectively, wherein the CRD2 domain alone has a high binding activity to the ligand. Therefore, we selected the CRD2 domain consisting of amino acid residues 68-110 of TACI to construct a fusion protein with the Fc portion of human antibody IgG1 (SEQ ID NO: 5) in the present application, thereby improving the expression disruption problem of TACI.

Because TACI proteins are prone to aggregation in neutral solutions, analysis of surface hydrophobic groups, charge groups, and sites of pharmaceutical risk (e.g., sites that pose a risk of aggregation) based on the crystal structure of TACI CRD2 (PDB ID:1XU 1) using Discovery studio software, showed potential risk of aggregation for TACI molecules at amino acids 69, 72, 73, 74, 77, 78, 85, 102, and/or 103. Meanwhile, we found that the protein structure of the TACI same family protein BCMA (6-42, PDB ID:1XU 2) has very high homology with the structure of the TACI, is basically consistent with the binding site of the ligand, and meanwhile, the N end part and the C end part of the TACI are not involved in the binding of the ligand (figure 1), more importantly, the analysis result of Discovery studio software shows that the BCMA has balanced surface charge distribution and aggregation risk is obviously lower than that of the TACI. Therefore, we designed a series of chimeric molecules of BCMA and TACI extracellular domains, and on the basis of retaining TACI and ligand binding sites, the N-terminal of TACI is replaced with the corresponding sequence of BCMA, and simultaneously the N-terminal and C-terminal of TACI are replaced with the corresponding sequence of BCMA and/or part of the pharmaceutical risk sites are subjected to amino acid replacement, so that the areas of the surface charge groups and hydrophobic groups of TACI proteins are reduced, and the aggregation risk of TACI is reduced. ALPN-303 (US 11274140B 2) and TELITACICEPT (RC-18, NC 101323643B) were also constructed as controls. The sequence of wild-type TACI (uniprot:O 14836, aa 1-293) is shown in SEQ ID NO. 1 in the present application. The sequence of wild-type BCMA (uniprot:Q 02223, aa 1-184) is shown in SEQ ID NO. 2 in the present application.

Construction of expression plasmid

The different TACI or TACI/BCMA chimeras were ligated to the Fc portion of human IgG1 via GSGGGGS (SEQ ID NO: 39) and constructed onto the pCDNA3.1 vector. In addition, ALPN-303 and TELITACICEPT (RC-18) for control were also constructed on the pCDNA3.1 vector for expression of the following proteins in Table 1:

TABLE 1 TACI or TACI/BCMA chimeric fusion proteins

The specific sequence information of the protein molecules is shown in a sequence table.

Expression purification of fusion proteins

Vectors containing the gene encoding the fusion protein were transferred into HEK293 cells using a chemical transfection method. Cultured HEK293 cells were transiently transfected using the chemical transfection reagent PEI according to the protocol provided by the manufacturer.

First, the expression plasmid DNA and the transfection reagent were prepared in an ultra clean bench, 5mL of Opti-MEM medium (Gibco cat# 31985-070) was added to a 50mL centrifuge tube, 50. Mu.g of the DNA corresponding to the plasmid was added, the Opti-MEM medium containing the plasmid was filtered using a 0.22 μm filter, followed by 150. Mu.g PEI (1 g/L) and allowed to stand for 20min. The DNA/PEI mixture was gently poured into 45mLHEK293 cells and mixed, incubated at 37℃in 8% CO ₂ for 20h, and then VPA was added to a final concentration of 2mM and 2% (v/v) Feed, followed by further incubation for 6 days.

After cell culture, the cell culture broth was centrifuged at 13000rpm for 20min, the supernatant was collected, and the supernatant was purified by a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). The procedure was as follows, the packed column was equilibrated with 5 column volumes of equilibration solution (PBS buffer (Gibco, cat# 70011-044)) prior to purification, the collected supernatant was passed through the column, the packed column was washed with 10 column volumes of equilibration solution to remove non-specific binding proteins, the packing was washed with 5 column volumes of elution buffer (100mM sodium citrate,pH 3.3), and the eluate was collected. mu.L of Tris (2M Tris) was added to each 1ml of eluate, and the eluate was exchanged into PBS buffer using ultrafiltration concentration tube (MILLIPORE, cat# UFC 901096), and the concentration was determined. 100. Mu.g of purified protein was taken, the concentration was adjusted to 1mg/mL, and the protein purity was measured using a gel filtration chromatography column SW3000 (TOSOH cat# 18675).

Purification results of TACI or TACI/BCMA chimeric fusion proteins and control samples are shown in table 2 below. The expression level of the TACI or the TACI/BCMA chimeric fusion protein in HEK293 cells is more than 100mg/mL, and the expression level of most chimeric fusion proteins is equivalent to that of a control sample. The purity of the TACI or TACI/BCMA chimeric fusion protein is more than 95 percent, which is equivalent to ALPN-303 and is obviously superior to RC-18.

TABLE 2 expression level and purity of TACI or TACI/BCMA chimeric fusion proteins and control samples

EXAMPLE 2 determination of affinity of TACI or TACI/BCMA chimeric fusion proteins to ligand

The equilibrium dissociation constant (KD) of the TACI or TACI/BCMA chimeric fusion proteins of the invention described above with their ligands was determined using a biological optical interferometry (ForteBio) assay.

ForteBio affinity assay the affinity of candidate molecules to BAFF and APRIL, respectively, was measured according to the existing method (Estep, P et al ,High throughput solution Based measurement ofantibody-antigen affinity and epitope binning.Mabs,2013.5(2):p.270-8): the sensor equilibrated for 20 minutes down the assay buffer centerline and then baseline was established for on-line detection for 120 seconds; forteBio affinity measurements were performed by loading candidate molecules onto an AHC sensor (Sartorius, 18-5060), the loaded candidate molecule sensors were placed in a solution containing 100nM BAFF or APRIL until the plateau phase, after which the sensor was transferred to assay buffer dissociation for at least 2 minutes for binding and dissociation rate measurement.

The experimental results are shown in Table 3. All TACI or TACI/BCMA chimeric fusion proteins showed significantly better affinity to BAFF and APRIL than the control samples, comparable to ALPN-303.

TABLE 3 affinity of TACI or TACI/BCMA chimeric fusion proteins with BAFF and APRIL

Example 3 in vitro function assay of TACI/BCMA chimeric fusion proteins

Example 3.1TACI/BCMA chimeric fusion proteins block binding of BAFF to IM-9 cells.

Samples (ALPN-303, RC-18 and TACI/BCMA chimeric fusion proteins) were subjected to a two-fold serial gradient dilution with an initial concentration of 200nM, three-fold dilution in FACS buffer, establishing a total of 12 concentration gradients. Biotin Human BAFF (Acro, cat#BAF82.2Q.sub.2-200 ug) was diluted to 0.2. Mu.g/mL, the diluted sample was mixed with the diluted Biotin human BAFF at 1:1, and incubated at 37℃for 30min. IM-9 cells (Nanjac, bai, # Cat CB 60276) were counted and diluted to 2X 10 ⁶/mL and 100. Mu.L/well was added to a U-bottom 96-well plate. The cell culture medium was removed by centrifugation at 500g for 5 min. The incubated samples were then added to the U-plate and the cells resuspended, 100. Mu.L/well, and incubated at 4℃for 30min. The supernatant was removed by centrifugation at 500g for 5min and the cells were washed 2 times with FACS buffer. 500g was centrifuged for 5min, FACS buffer was removed, 50. Mu. LPE STREPTAVIDIN secondary antibody (BD, cat# 554061) was added to each well (1:200 dilution in FACS buffer), and incubated at 4℃for 30min in the dark. The supernatant was removed by centrifugation at 500g for 5min and the cells were washed 3 times with FACS buffer. Cells were resuspended with 200. Mu. LFACS buffer and examined by flow cytometry.

The results show that ALPN-303, RC-18, and TACI/BCMA chimeric fusion proteins all inhibit BAFF binding to IM-9 cells. However, TACI/BCMA chimeric fusion proteins were significantly better than RC-18 in inhibiting BAFF binding to IM-9 cells (IC 50 of 12.47+ -3.73 nM) and comparable to ALPN-303 in inhibiting activity (IC 50 of 1.72+ -0.48 nM) (Table 4).

Example 3.2TACI/BCMA chimeric fusion proteins block binding of APRIL to IM-9 cells.

Samples (ALPN-303, RC-18 and TACI/BCMA chimeric fusion proteins) were serially diluted three times at an initial concentration of 200nM, three times in FACS buffer, and a total of 12 concentration gradients were established. Biotin Human APRIL (Acro, cat#APL-H82F5-200 UG) was diluted to 0.2. Mu.g/mL, diluted samples were mixed with diluted Biotin human APRIL at 1:1, and incubated at 37℃for 30min. IM-9 cells were counted and diluted to 2X 10 ⁶/mL and 100. Mu.L/well was added to the U-bottom 96-well plate. The cell culture medium was removed by centrifugation at 500g for 5 min. The incubated samples were then added to the U-plate and the cells resuspended, 100. Mu.L/well, and incubated at 4℃for 30min. The supernatant was removed by centrifugation at 500g for 5min and the cells were washed 2 times with FACS buffer. 500g was centrifuged for 5min, FACS buffer was removed, 50. Mu. LPE STREPTAVIDIN secondary antibody (BD, cat# 554061) was added to each well (1:200 dilution in FACS buffer), and incubated at 4℃for 30min in the dark. The supernatant was removed by centrifugation at 500g for 5min and the cells were washed 3 times with FACS buffer. Cells were resuspended with 200. Mu. LFACS buffer and examined by flow cytometry.

The results showed that ALPN-303, RC-18, and TACI/BCMA chimeric fusion proteins all inhibited APRIL binding to IM-9 cells. However, TACI/BCMA chimeric fusion proteins were significantly better than RC-18 in inhibiting the binding of APRIL to IM-9 cells (IC 50 of 5.89.+ -. 2.92 nM) and comparable to ALPN-303 in inhibiting activity (IC 50 of 0.35.+ -. 0.29 nM) (Table 4).

Example 3.3 blocking of the NF-. Kappa. B ReporterJurkat Signal by the TACI/BCMA chimeric fusion protein by BAFF/APRIL-induced surface overexpression of human TACI

The cDNA encoding human TACI was cloned into the pLenti-IRES-puro vector (Invitrogen) and then NF- κB Jurkat report cells (Gift Biol., cat#GM-C07855) were transfected by lentivirus to generate NF- κ BReporter Jurkat cells overexpressing human TACI (i.e., TACI/NF- κ B Reporter Jurkat cells). The chimeric TACI/BCMA-Fc fusion protein was then tested for blocking the BAFF/APRIL-induced TACI/NF- κ B Reporter Jurkat signaling using a gradient dilution method.

Briefly, samples (ALPN-303, RC-18 and TACI/BCMA chimeric fusion proteins) were subjected to three-fold serial dilutions, starting at 1200nM, three-fold dilutions in RPMI-1640+10% FBS medium, establishing a total of 10 concentration gradients. The Human BAFF (Acro, cat#BAFH2D 4) + HumanAPRIL (Acro, cat#APL-H52D 1) was diluted to 20nm using RPMI-1640+10% FBS medium, the diluted samples were mixed with diluted 20nm Human BAFF/APRILmixture at 1:1 and incubated at 37℃for 30min. TACI/NF- κ B Reporter Jurkat cells were counted and diluted to 1X 10 ⁶ cells/mL and 50. Mu.L/well was added to a white flat bottom 96-well plate. The incubated samples were then added to a white flat bottom 96-well plate containing cells and resuspended, incubated at 50. Mu.L/well for 22h at 37℃and after incubation, the cells were assayed for luminescent signals using Bio-Lite LuciferaseAssay System (Vazyme, #DD 1201-03).

The results show that ALPN-303, RC-18, and TACI/BCMA chimeric fusion proteins each inhibit the BAFF/APRIL binding to TACI induced NF- κ B Reporter Jurkat signaling. However, the inhibitory activity of TACI/BCMA chimeric fusion protein against the NF- κ B Reporter Jurkat signal induced by BAFF/APRIL binding to TACI was significantly better than that of RC-18 (IC 50: 23.55.+ -. 8.47 nM) and comparable to ALPN-303 (IC 50: 6.40.+ -. 2.02 nM) (Table 4).

Example 3.4 blocking of NF- κ B ReporterJurkat signaling by TACI/BCMA chimeric fusion proteins by BAFF/APRIL-induced surface over-expression of human BCMA

The cDNA encoding human BCMA was cloned into the pLenti-IRES-Neo vector (Invitrogen) and then NF- κB Jurkat report cells (Ji Mannheim, cat#GM-C07855) were transfected by lentivirus to generate NF- κ B Reporter Jurkat cells overexpressing human BCMA (i.e., BCMA/NF- κ B Reporter Jurkat cells). The chimeric TACI/BCMA-Fc fusion protein was then tested for blocking BAFF/APRIL-induced BCMA/NF- κ B Reporter Jurkat signaling using a gradient dilution method.

Briefly, samples (ALPN-303, RC-18 and TACI/BCMA chimeric fusion proteins) were serially diluted three-fold at an initial concentration of 2400nM, three-fold in RPMI-1640+10% FBS medium, and a total of 10 concentration gradients were established. The Human BAFF (Acro, cat#BAFH2D 4) + HumanAPRIL (Acro, cat#APL-H52D 1) was diluted to 20nm using RPMI-1640+10% FBS medium, the diluted samples were mixed with diluted 20nm Human BAFF/APRILmixture at 1:1 and incubated at 37℃for 30min. BCMA/NF- κ B Reporter Jurkat cells were counted and diluted to 2X 10 ⁶ cells/mL and 50. Mu.L/well was added to a white flat bottom 96-well plate. The incubated sample was then added to a white flat bottom 96-well plate containing cells and resuspended, and incubated at 50. Mu.L/well for 5h at 37℃and after incubation, the cells were assayed for luminescent signals using Bio-Lite LuciferaseAssay System (Vazyme, #DD 1201-03).

The results show that ALPN-303, RC-18, and TACI/BCMA chimeric fusion proteins all inhibit BAFF/APRIL binding to BCMA-induced NF- κ B Reporter Jurkat signaling. However, the TACI/BCMA chimeric fusion protein inhibited the BAFF/APRIL binding to BCMA-induced NF- κ B Reporter Jurkat signaling significantly better than RC-18 (IC 50 of 53.25.+ -. 8.81 nM) and comparable to ALPN-303 (IC 50 of 4.06.+ -. 0.53 nM) (Table 4).

Example 3.5 detection of ability of TACI/BCMA chimeric fusion proteins to block proliferation of spleen cells

Spleen cells from mice were lysed, cell density was adjusted to 2X 10 ⁶/mL, 50. Mu.L per well was added and cells plated at 1X10 ⁵/well. Samples (ALPN-303, RC-18 and TACI/BCMA chimeric fusion proteins) were subjected to three-fold serial dilutions, starting at 1200nM, three-fold dilutions in RPMI-1640+10% FBS medium, establishing a total of 9 concentration gradients. The Human BAFF (Acro, cat#BAFH2D 4) was diluted to 50ng/mL using RPMI-1640+10% FBS medium, the diluted samples were mixed with diluted Human BAFF at 1:1 and incubated at 37℃for 30min.

Control wells were set up in the IgG group (using IgG (Equitech-Bio, cat#SLH56) as sample+cells+BAFF antigen) and in the blank group (RPMI-1640+10% FBS) (medium+cells).

50. Mu.L/Kong Fuyo of the mixture was added to the cells, incubated at 37℃for 72h, after which the proliferation of the cells was detected using CellCount-LiteTM Luminescent Cell Viabil (Vazyme, #DD 1101-02).

IC50 was calculated by curve fitting based on readings at different concentrations. The results showed that ALPN-303, RC-18, and TACI/BCMA chimeric fusion proteins all inhibited BAFF-induced spleen cell proliferation. However, TACI/BCMA chimeric fusion proteins were significantly superior to RC-18 in inhibiting BAFF-induced spleen cell proliferation (IC 50 of 4.98.+ -. 0.95 nM), comparable to ALPN-303 in inhibiting activity (IC 50 of 0.49.+ -. 0.06 nM) (Table 4).

TABLE 4 in vitro functional Activity of TACI/BCMA chimeric fusion proteins

Example 3.6 detection of ability of TACI/BCMA chimeric fusion proteins to inhibit B cell proliferation

PBMC (AllCell, cat#FPB004F-C) were recovered, B cells were isolated using EASYSEPTM HUMAN B CELL ENRICHMENT KIT (STEMCELL, cat# 19054), cell densities were counted and adjusted to 2X 10 ⁶/mL, and rhIL-4 (R & D, cat#204-IL-050) and Anti-human IgM (Jakson, cat#109-006-129) were added at a final concentration of 20 ng/mL. Samples (ALPN-303, RC-18 and TACI/BCMA chimeric fusion proteins) were serially diluted three times at an initial concentration of 2400nM, three times in RPMI-1640+10% FBS medium, and 9 concentration gradients were established in total.

Human BAFF (Acro, cat#BAFH2D 4) + HumanAPRIL (Acro, cat#APL-H52D 1) was diluted to 20nm using RPMI-1640+10% FBS medium, diluted samples were mixed with diluted 20: 20nm humanBAFF/APRIL mix at 1:1, and incubated at 37℃for 30min. Control wells were set up, igG group (using IgG (Equitech-Bio, cat#SLH56) as sample), 50. Mu.L/Kong Fuyo of the mixture was added to the cells, incubated at 37℃for 72h, and after 72h incubation cells were examined for proliferation and dose-response curves using CellCount-LiteTM Luminescent Cell Viabil (Vazyme, # DD 1101-02).

The results showed that ALPN-303 and TACI/BCMA chimeric fusion proteins were both effective in inhibiting BAFF/APRIL-induced B cell proliferation (FIG. 4). The inhibition ratio in the figure is calculated as experimental group/control IgG group multiplied by 100%.

EXAMPLE 4 stability assay of TACI/BCMA chimeric fusion proteins

The thermostability of TACI/BCMA chimeric fusion proteins was assessed by Dynamic Light Scattering (DLS), differential Scanning Calorimetry (DSC) and accelerated stability.

Dynamic Light Scattering (DLS) assessment of thermal stability

Centrifuging protein (1 mg/mL) solution at 13000g/min for 5min, adding into a sample plate, detecting the change of the particle size of protein molecules in the continuous temperature rising process, setting the detection as DLS acquisition time of 5s, acquisition times of 5 times and experimental temperature of 25-85 ℃. The change of the particle size of the sample with temperature was analyzed after the end of the experiment.

Differential Scanning Calorimetry (DSC) evaluation of thermal stability

The antibody mutant sample was diluted to 0.5-1mg/mL with PBS solution, the diluted sample and PBS buffer were degassed, then the sample was added to the left sample plate, and PBS buffer was added at the corresponding position in the right reference plate. The initial temperature is 30 ℃ and the equilibrium time is 10min, and the heating is at a rate of 1 ℃ per min, and the final temperature is 90 ℃.

The results of the DLS and DSC experiments are shown in Table 5. Experimental results show that the initial aggregation temperature and the protein denaturation temperatures T _m and T _m 2 of the TACI/BCMA chimeric fusion proteins 99017 and 99018 are significantly better than those of the control RC18 and unmodified TACI CRD2 molecule 99001, and the thermal stability is equivalent to those of ALPN-303.

TABLE 5 patent drug Properties of TACI/BCMA chimeric fusion proteins

Accelerated stability assessment of TACI/BCMA chimeric fusion proteins

In addition, the TACI/BCMA chimeric fusion protein was replaced with an ultrafiltration tube into PBS solution at pH7.4 at a concentration of 10mg/ml, and subjected to an acceleration stability test in a constant temperature incubator at 50℃for 4 days, and sampled for purity (SEC and CE-SDS method) analysis. The experimental results are shown in Table 6.

The experimental results show that the stability of TACI/BCMA chimeric fusion proteins 099017 and 099018 is best under conditions of 50 ℃ for 4 days, the protein SEC purity can be maintained above 95%, and the non-reducing CE-SDS purity can be maintained above 98%. Stability was significantly better than control RC-18 and unmodified TACI CRD2 molecule 99001, as well as control ALPN-303.

TABLE 6 evaluation of forced stability of TACI/BCMA chimeric fusion proteins by CE-SDS

EXAMPLE 5 in vivo efficacy of TACI/BCMA chimeric fusion proteins

EXAMPLE 5.1 efficacy experiment of TACI/BCMA chimeric fusion proteins in mouse KLH immune model

C57BL/6N mice (available from Beijing Vitre Liwa laboratory animal technologies Co., ltd.) were selected and injected intraperitoneally with 0.2mg KLH (Solarbiol, cat#KB160) containing Alum adjuvant (Thermo, cat# 77161) on days 0, 12. The experiments were performed on 5 groups of Blank (Blank, no treatment), model control IgG group, 99001 group, 99006 group and 99017 group, 5-7 mice each (3 mice of Blank control group), and on days 4 and 11 by intraperitoneal administration (model control IgG group injected with an equivalent amount of IgG antibody (purchased from Equitech-Bio, cat#slh56)), on day 20 mice spleens were taken for cell detection B cell changes and blood was collected for detection of IgA (Invitrogen, cat# EMIGA), igM (Invitrogen, cat#88-50470-88) and IgG (Invitrogen, cat#88-50400-88) in serum.

The results showed that both the dosing groups inhibited proliferation of spleen cells, B cells in the spleen (table 7), and IgA, igM, and IgG levels in serum (table 8) resulting from KLH immunization compared to the control IgG group. The results of calculation of the inhibition ratio of the administration group to the IgG group show that the inhibition effect of 99017 group (TACI/BCMA chimeric fusion protein) is superior to that of 99001 group (unmodified TACI CRD2 molecule) and 99006 group (unmodified BCMACRD molecule). The above data suggest that engineered TACI/BCMA chimeric fusion proteins are significantly better for B cells and antibody levels than TACI or BCMAWT CRD fragments.

TABLE 7 number of spleen cells and B cells in spleen (n=5-7 cells/group)

^* Represents a significant difference in 99017 compared to 99001 (^** p <0.01, ttest).

^# Represents 99017 was significantly different from 99006 (^## p <0.01, t test).

Table 8 serum levels of IgA, igM and IgG (n=5-7/group)

^# Represents 99017 was significantly different from 99006 (^### p <0.001, t test).

EXAMPLE 5.2 efficacy of TACI/BCMA chimeric fusion proteins in murine BAFF stimulation models

Balb/c mice (available from Beijing Veityle Liwa laboratory animal technologies Co., ltd.) were selected and injected intraperitoneally with 10. Mu.g of human BAFF protein (Sino Biological, cat#10056-HNCHA) on days 0, 1,2, and 3. The experiments were performed with a Blank (Blank, without any treatment), a model control IgG group, RC-18, ALPN-303 and TACI/BCMA chimeric fusion protein groups, 5 groups of 5-7 mice each (3 mice in the Blank), and with intraperitoneal administration on days 0 and 2 (model control IgG group injected with an equivalent amount of IgG antibody (from Equitech-Bio, cat#SLH56)), spleens of mice were taken on day 4 for cell detection on serum IgA (Invitrogen, cat# EMIGA), igM (Invitrogen, cat#88-50470-88) and IgG (Invitrogen, cat#88-50400-88) using ELISA kits. The results showed that TACI/BCMA chimeric fusion protein was effective in inhibiting BAFF-induced proliferation of spleen cells and B cells in spleen (table 9), while TACI/BCMA chimeric fusion protein was effective in reducing BAFF-induced serum IgA, igM and IgG levels (table 10).

TABLE 9 number of spleen cells and B cells in spleen (n=5-7/group)

Table 10 serum levels of IgA, igM and IgG (n=5-7/group)

EXAMPLE 5.3 efficacy experiment of TACI/BCMA chimeric fusion proteins in mouse KLH immune model

C57BL/6N mice (available from Beijing Vitre Liwa laboratory animal technologies Co., ltd.) were selected and injected intraperitoneally with 0.2mg KLH (Solarbiol, cat#KB160) containing Alum adjuvant (Thermo, cat# 77161) on days 0, 12. The experiments set up a placebo, model control IgG, RC-18, ALPN-303 and TACI/BCMA chimeric fusion protein (99017 and 99018, respectively) for 5 groups of 5-7 mice each (3 mice in the placebo group), and were given by intraperitoneal injection on days 4 and 11 (model control IgG was given an equivalent amount of IgG antibody (from Equitech-Bio, cat#slh56)), spleens from mice were taken on day 20 for cell detection and blood was collected for detection of IgA (Invitrogen, cat# EMIGA), igM (Invitrogen, cat#88-50470-88) and IgG (Invitrogen, cat#88-50400-88) in serum.

The results showed that the administration group inhibited proliferation of spleen cells, B cells in spleen (fig. 5), and different B cell subsets generated by KLH immunization (table 11) compared to the control IgG group. However, compared with RC-18 group, the TACI/BCMA chimeric fusion protein group has better inhibition effect, and compared with ALPN-303, the TACI/BCMA chimeric fusion protein can inhibit proliferation of MZ cells, GC B cells and plasma cells more effectively. Meanwhile, TACI/BCMA chimeric fusion proteins were effective in reducing the IgA, igM and IgG levels in serum generated by KLH immunization (fig. 6, table 12).

TABLE 11 proportion of different B cell subsets to total spleen cells (n=5-7/group)

^* Represents 99017 was significantly different from ALPN-303 (^*p<0.05,^** p <0.01, t test).

Table 12 serum levels of IgA, igM and IgG (n=5-7/group)

EXAMPLE 5.4 pharmacokinetic Studies of TACI/BCMA chimeric fusion proteins in mice

Mice were used for intravenous injection (Intravenous Injection, i.v.) to examine the pharmacokinetics of the chimeric fusion proteins. Each group of 9 BALB/c mice has a weight of about 20g, each mouse is subjected to intravenous injection of 10mg/kg of molecules to be detected, blood is collected from the eye sockets 5min, 0.5h, 2h, 6h, 2 nd day, 4 th day, 7 th day, 14 th day and 21 th day after single administration, and blood is naturally coagulated and centrifuged to obtain serum.

The concentration of antibody drug in serum was determined by dissolving a coating of carbonate powder (Thermo, cat# 23282) in 400mL of ultra pure water, mixing well, metering to 500 mL), diluting antigen humanAPRIL-his protein (Acro Biosystems, cat# APL-H52D 1) to 1 μg/mL, adding 100 μl per well to 96-well ELISA plate (Thermo, cat# 442404), and incubating overnight at 4 ℃. The coating solution was discarded, and 1 XPBST was washed 3 times. mu.L of blocking solution (PBST solution containing 2% BSA) was added to each well and blocked at room temperature for 1h. After 3 washes of 1 XPBST, the blocking solution was discarded, diluted mouse serum was added and incubated for 2 hours at room temperature. The solution in the ELISA plate was discarded and washed 5 times with 1 XPBST. Diluted Goat x-human IgG-Fc-HRP (BETHYL, cat#A80-104P) was added and incubated for 1 hour at room temperature at 100. Mu.L per well. The solution in the ELISA plate was discarded and washed 5 times with 1 XPBST. 100 mu LTMB of color development solution (Soy palett, cat#PR 1200) was added to each well and developed for 5-10min, and 50 mu L of stop solution (Soy palett, cat#C1058) was added to each well to terminate. The microplate reader reads OD450nm and OD620nm values. The change in plasma concentration at various time points in mice given the test molecule (10 mg/kg to mice) is shown in FIG. 7. The pharmacokinetic parameters were calculated using an Excel PK solver calculation non-compartmental model, with the primary drug exposure parameters (T1/2, cmax, AUC0-T, AUC0- ≡, CL and Vss) as shown in Table 13. By analyzing the mouse pharmacokinetic parameters of the different molecules, the pharmacokinetics of the TACI/BCMA chimeric fusion protein was significantly better than RC-18 and slightly better than ALPN-303.

TABLE 13 mouse pharmacokinetic parameters of TACI/BCMA chimeric fusion proteins

EXAMPLE 6 toxicological kinetics Studies of TACI/BCMA chimeric fusion proteins on cynomolgus monkeys

The TK of the 99017 molecule was detected by subcutaneous injection (Subcutaneous Injection, s.c.) using cynomolgus monkeys. Each dose group of 2 cynomolgus monkeys (1 female and 1 male) has a weight of about 3.5-4.5kg, each group of cynomolgus monkeys is subcutaneously injected with 100mg/kg or 200mg/kg of molecules to be detected every week for five times, blood collection is started after 24 hours (C1D 1) after the first administration and 24 hours (C4D 1) after the fourth administration respectively, blood collection time points are 30min, 2 hours, 6 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours and 168 hours, and serum is collected after the blood is naturally coagulated.

The concentration of antibody drug in serum was determined by diluting antigen humanAPRIL-his protein (Acro Biosystems, cat#APL-H52D 1) to 2. Mu.g/mL with coating solution (a solution of a carbonate powder in 400mL of ultra pure water, mixed well to a volume of 500 mL), adding 100. Mu.L per well to 96-well ELISA plate, and incubating at 4℃overnight. The coating solution was discarded, and 1 XPBST was washed 3 times. mu.L of blocking solution (containing 5% SM-1 in PBST) was added to each well and blocked at room temperature for 2h. After 3 washes of 1 XPBST, 100. Mu.L of diluted cynomolgus monkey serum was added to each well and incubated for 2 hours at room temperature. The solution in the ELISA plate was discarded and washed 3 times with 1 XPBST. Diluted Human IGG HEAVY AND LIGHT CHAIN Monkey-AdsorbedAntibody (BETHYL, cat#A80-319P) was added and incubated for 1 hour at room temperature at 100. Mu.L per well. The solution in the ELISA plate was discarded and washed 5 times with 1 XPBST. 100 mu LTMB of color development solution is added to each well, the color development is carried out for 5-10min, and 100 mu L of stop solution is added to each well to stop. The microplate reader reads OD450nm and OD620nm values. The change in blood concentration at different time points after administration of different doses of the molecules to be tested to cynomolgus monkeys at C1D1 and C4D1 is shown in FIG. 8. Relevant kinetic parameters were calculated using PhoenixWinNonlin 8.4.4 non-compartmental model, with the primary drug exposure parameters (AUC 0-t, cmax, tmax, T _1/2, CI/F) as shown in Table 14.

The results are shown in FIG. 8, where 200mg/kg 99017 has a kinetic profile of greater than 100mg/kg at C1D1 and C4D1, and is dose dependent. The kinetic parameters of the calculated different doses after different dosing frequency were analyzed, with 100mg/kg of T _1/2 being approximately 5-5.9 days, and 150mg/kg of T _1/2 disclosed in ALPN-303 being 2.9 days (Dillon S,Evans L,Lewis K,et al.ALPN-303,an Enhanced,Potent Dual BAFF/APRIL Antagonist Engineered by Directed Evolution for the Treatment of Systemic Lupus Erythematosus(SLE)and Other B Cell-Related Diseases.Arthritis Rheumatol.2021;73(suppl 9);https://www.alpineimmunesciences.com/wp-content/uploads/2021/12/ALPN-303_ACR-2021.pdf)., thus the half-life of 99017 was significantly better than ALPN-303.

TABLE 14 Macaca fascicularis toxicological parameters

EXAMPLE 7 pharmacokinetic and pharmacodynamic studies of TACI/BCMA chimeric fusion proteins and YTE molecules on cynomolgus monkeys

Experiments were performed by intravenous injection (Intravenous Injection, i.v.) using cynomolgus monkeys to examine the pharmacokinetics and pharmacodynamics of the 99017 and 99025 molecules. Each group of 3 cynomolgus monkeys has a weight of about 3.5-4.5kg, each cynomolgus monkey is subjected to intravenous injection of 9mg/kg of molecules to be detected, blood samples are collected after single administration for 5min, 2h, 6h, 12h, 24h, 72h, 144h, 216h, 312h, 432h, 480h, 624h, 816h and 984h, and blood is naturally coagulated, and serum is collected by centrifugation for detecting the concentration of antibody drugs in serum. Blood samples were collected before and after single dosing, 6d, 9d, 12d, 18d, 24d, 32d and 40d, respectively, blood was naturally coagulated, serum was collected by centrifugation, igA (Cat# 05219205190), igM (Cat# 05220726190) and IgG (Cat# 05220718190) in serum were detected using the Roche Diagnostic immunoglobulin detection kit, and the percentage of immunoglobulin decrease in serum was calculated at different time points after and before dosing, and the results are shown in FIG. 10. The results show that TACI/BCMA chimeric fusion proteins were effective in reducing BAFF-induced IgA, igM and IgG levels in serum.

The concentration of antibody drug in serum was determined by diluting antigen humanAPRIL-his protein (Acro Biosystems, cat#APL-H52D 1) to 2. Mu.g/mL with coating solution (a solution of a carbonate powder in 400mL of ultra pure water, mixed well to a volume of 500 mL), adding 100. Mu.L per well to 96-well ELISA plate, and incubating at 4℃overnight. The coating solution was discarded, and 1 XPBST was washed 3 times. mu.L of blocking solution (containing 5% SM-1 in PBST) was added to each well and blocked at room temperature for 2h. After 3 washes of 1 XPBST, 100. Mu.L of diluted cynomolgus monkey serum was added to each well and incubated for 2 hours at room temperature. The solution in the ELISA plate was discarded and washed 3 times with 1 XPBST. Diluted Human IGG HEAVY AND LIGHT CHAIN Monkey-AdsorbedAntibody (BETHYL, cat#A80-319P) was added and incubated for 1 hour at room temperature at 100. Mu.L per well. The solution in the ELISA plate was discarded and washed 5 times with 1 XPBST. 100 mu LTMB of color development solution is added to each well, the color development is carried out for 5-10min, and 100 mu L of stop solution is added to each well to stop. The microplate reader reads OD450nm and OD620nm values. The change in blood concentration of the cynomolgus monkey to which the test molecule was administered (administered to the cynomolgus monkey at 9 mg/kg) at various time points is shown in fig. 9.

The results are shown in figures 9 and 10, with the 99025 pharmacokinetics being longer and the pharmacodynamics being better than 99017.

While Cmax and AUC were better for (Dillon S,Evans L,Lewis K,et al.ALPN-303,an Enhanced,Potent Dual BAFF/APRIL Antagonist Engineered by Directed Evolution for the Treatment of Systemic Lupus Erythematosus(SLE)and Other B Cell-Related Diseases.Arthritis Rheumatol.2021;73(suppl 9);Lawrence S.Evans,Katherine E.Lewis,Daniel DeMonte,et al.Povetacicept,an Enhanced Dual APRIL/BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell–Related Autoimmune Diseases.https://doi.org/10.1002/art.42462),99017 and 99025 than the data disclosed in ALPN-303 (table 15), the pharmacodynamic retention time was longer.

Table 15 comparison of cynomolgus monkey pharmacokinetic Cmax and AUC

And (3) a sequence table:

Claims

1. A TACI/BCMA chimera comprising a chimeric protein in which the N-terminus and/or C-terminus of a functional fragment of the extracellular domain ECD of TACI is replaced by the N-terminus and/or C-terminus of BCMA.

2. The TACI/BCMA chimera of claim 1, comprising the following structure:

BCMAN terminal amino acid-TACI portion, wherein the TACI portion is the extracellular domain ECD of TACI lacking the N-terminus or a functional fragment thereof, or

BCMAN terminal amino acid-TACI part-BCMAC terminal amino acid, wherein the TACI part is the extracellular domain ECD of TACI lacking the N-terminus and C-terminus or a functional fragment thereof.

3. The TACI/BCMA chimera of claim 1 or 2, wherein the functional fragment of the extracellular region ECD of TACI comprises CRD2 of TACI and does not comprise CRD1 or any fragment of CRD1;

Preferably, the CRD2 is the amino acid sequence of TACI corresponding to positions 71-104 shown in SEQ ID NO: 1, and the CRD1 is the amino acid sequence of TACI corresponding to positions 34-66 shown in SEQ ID NO: 1;

Optionally, the extracellular ECD functional fragment of TACI further comprises a partial stem region of TACI and/or any amino acid sequence corresponding to positions 68-70 of SEQ ID NO: 1;

Preferably, the functional fragment of the extracellular region ECD of TACI is or comprises the following fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO: 1: amino acid residues 68-110; for example,

The extracellular domain ECD functional fragment of TACI comprises

(i) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 31, 32 or 33;

(ii) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33 and has an amino acid sequence of Y102D;

(iii) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32 and has K77E, F78Y and Y102D;

(iv) the amino acid sequence shown in SEQ ID NO: 31, 32 or 33; or

(v) consisting of the sequence of any one of (i) to (iv).

4. The TACI/BCMA chimera of any one of claims 1 to 3, wherein the N-terminus of the functional fragment of the extracellular domain ECD of TACI refers to the N-terminal amino acid of TACI preceding amino acid Y79 of SEQ ID NO: 1; and/or

The C-terminus of the extracellular ECD functional fragment of TACI refers to the C-terminal amino acid after the 99th amino acid, the 100th amino acid, the 101st amino acid, the 102nd amino acid, the 103rd amino acid, the 104th amino acid, the 105th amino acid, the 106th amino acid, the 107th amino acid, the 108th amino acid, the 109th amino acid or the 110th amino acid of TACI corresponding to SEQ ID NO: 1, preferably the C-terminal amino acid after the 99th or 105th amino acid.

5. The TACI/BCMA chimera of any one of claims 1 to 4, wherein the N-terminal amino acids of BCMA are selected from the amino acid sequence of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, or 7-13 of SEQ ID NO: 2; preferably, the N-terminal amino acid sequence of BCMA comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 40-46; and/or

The C-terminal amino acid sequence of BCMA is the amino acid sequence of BCMA corresponding to positions 37-47, 37-46, 37-45, or 37-44 of SEQ ID NO: 2, or the amino acid sequence corresponding to positions 43-44 or 43-46 of SEQ ID NO: 2;

Optionally, the C-terminal amino acid of BCMA comprises a mutation, such as a substitution, that improves binding affinity, increases stability and/or improves druggability, for example, the mutation is a mutation at position 39 and/or position 42, such as a substitution, such as N42A or N42Q or R39D or N42A-R39D;

Preferably, the C-terminal amino acid sequence of BCMA comprises the amino acid sequence shown in any one of SEQ ID NOs: 47-55, or consists of the amino acid sequence.

6. The TACI/BCMA chimera of any one of claims 1 to 5, wherein the TACI ECD functional fragment comprises an amino acid site mutation that reduces aggregation risk, such as a mutation such as a substitution at a druggability risk site, for example, the druggability risk site is selected from amino acids 69, 72, 73, 74, 77, 85, 102, or 103 corresponding to SEQ ID NO: 1, preferably, the amino acid at the druggability risk site is mutated to A or D, for example, the mutation is Y102D;

Preferably, the TACI portion comprises the amino acid sequence shown in any one of SEQ ID NOs: 35, 31-34 or 36-38, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical thereto, or consists of the amino acid sequence.

7. The TACI/BCMA chimera of any one of claims 1-6, wherein the TACI/BCMA chimera comprises the amino acid sequence set forth in any one of SEQ ID NOs: 73, 59-72, or 74-80, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical thereto.

8. A fusion protein comprising the TACI/BCMA chimera according to any one of claims 1 to 7, and an Fc region;

Preferably, the Fc region is human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc;

Optionally, the Fc region comprises one or more of the following mutations:

(i) Lysine K deleted at the C-terminus (K447del);

(ii) mutations that reduce effector function mediated by the Fc region;

(iii) mutations that reduce binding to Fcγ receptors, such as L234A/L235A mutations or L234A/L235E or L234A/L235E/G237A mutations;

(iv) mutations that enhance the binding of the Fc fragment to FcRn, such as M252Y/S254T/T256E and/or M428L/N434S;

For example, the Fc region comprises

(i) the amino acid sequence of SEQ ID NO: 81 or 82, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical thereto;

(ii) the amino acid sequence shown in SEQ ID NO: 83 or 84, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto, optionally lacking the C-terminal lysine;

(iii) an amino acid sequence as set forth in any one of SEQ ID NOs: 85-88 and 100-103, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 98%, 99% or more identity thereto;

(iv) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 85 or 87 and comprises the mutations L234A/L235E/G237A;

(v) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 86 or 88 and comprises the mutations L234A/L235E/G237A and a C-terminal deleted lysine;

(vi) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 100 or 102 and comprises the mutations L234A/L235E/G237A and M252Y/S254T/T256E;

(vii) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 101 or 103 and comprises the mutations L234A/L235E/G237A, M252Y/S254T/T256E, and a C-terminal deleted lysine; or

(viii) consisting of the amino acid sequence described in any one of (i) to (vii) above.

9. The fusion protein of claim 8, wherein the TACI/BCMA chimera is fused to Fc directly or through a linker, preferably, the C-terminus of the chimera is fused to the N-terminus of Fc directly or through a linker;

Optionally, the linker is selected from (GSGGGGS) _n , (GS) _n , (GSGGS) _n , (GGGGS) _n or (GGGS) _n , wherein n is an integer of at least 1, such as 1, 2, 3, 4 or 5; for example, (GSGGGGS) _n , wherein n=1-3, for example, the linker is the amino acid sequence shown in SEQ ID NO:39.

10. The fusion protein of claim 8 or 9, wherein the fusion protein

(i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 10, 5-9 and 11-28; or

(ii) comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 10, 5-9, and 11-28.

A fusion protein dimer comprising a first monomer and a second monomer, wherein the first and second monomers respectively comprise the fusion protein chain according to any one of claims 8 to 10, preferably the first monomer and the second monomer are identical.

12. A polynucleotide encoding the TACI/BCMA chimera according to any one of claims 1 to 7, the fusion protein according to any one of claims 8 to 10, or the fusion protein dimer according to claim 11.

13. An expression vector comprising the polynucleotide according to claim 12, for example, the expression vector is a pCDNA expression vector, for example, a pCDNA3.1 expression vector.

14. A host cell comprising the polynucleotide of claim 12 or the expression vector of claim 13.

15. A method for preparing a TACI/BCMA chimera or a fusion protein or fusion protein dimer thereof, wherein the method comprises culturing the host cell of claim 14 under conditions suitable for the expression of the TACI/BCMA chimera or a fusion protein or fusion protein dimer thereof, and optionally recovering the TACI/BCMA chimera or a fusion protein or fusion protein dimer thereof from the host cell (or host cell culture medium).

16. A pharmaceutical composition comprising the TACI/BCMA chimera according to any one of claims 1 to 7, the fusion protein according to any one of claims 8 to 10, or the fusion protein dimer according to claim 11, and optionally a pharmaceutically acceptable excipient.

17. A drug combination comprising the TACI/BCMA chimera of any one of claims 1 to 7, the fusion protein of any one of claims 8 to 10, or the fusion protein dimer of claim 11, and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation), antibodies or small molecule drugs, or immunomodulators (e.g., immunosuppressants)).

18. A method for preventing or treating a disease in a subject, such as a B cell or autoantibody-related disease or an immune system disease (such as an autoimmune disease) or inflammation, the method comprising administering to the subject the TACI/BCMA chimera of any one of claims 1 to 7, the fusion protein of any one of claims 8 to 10, the fusion protein dimer of claim 11, the pharmaceutical composition of claim 16, or the pharmaceutical combination of claim 17.

For example, the B cell or autoantibody-related disease or immune system disease is an autoimmune disease mediated by B cells or autoantibodies;

Optionally, the B cell or autoantibody-related disease or immune system disease or inflammation is a disease in which B cells in an individual abnormally proliferate or activate compared to a sample of a healthy individual, such as an autoimmune disease;

Preferably, the disease is lupus such as systemic lupus erythematosus, rheumatoid arthritis, chronic kidney disease such as IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart disease (CAD) or thyroid eye disease;

Optionally, the administration further comprises co-administration of one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).